EA040134B1 - METHOD FOR OBTAINING FOOD COMPOSITION CONTAINING BETA-LACTOGLOBULIN IN CRYSTALIZED FORM - Google Patents

Description

The field of technology to which the invention belongs

The invention relates to a new method for preparing compositions based on isolated beta-lactoglobulin and/or compositions containing crystallized beta-lactoglobulin. In addition, the present invention relates to new compositions based on beta-lactoglobulin, to options for the use of these compositions and to food products containing these compositions.

Prerequisites for the invention

The concept of milk protein fractionation is well known in the art and has been developed over the past decades for a number of technologies in order to obtain compositions enriched with various types of milk proteins, each with specific properties and characteristics.

The isolation of beta-lactoglobulin (BLG) from whey has been the subject of a number of publications and typically involves multiple separation steps and often chromatographic techniques to obtain a purified beta-lactoglobulin containing product.

For example, de Jongh et al. (Mild Isolation Procedure Discloses New Protein Structural Properties of (3Lactoglobulin, J. Dairy Sci., vol. 84(3), 2001, pages 562-571) describes the purification of BLG from fresh milk by low temperature acid coagulation of casein and by acting on obtained acidic serum by a combination of affinity chromatography (DEAE Sepharose) and gel permeation chromatography The resulting BLG composition was noted to contain 0.985 g of beta-lactoglobulin per 1 g of protein.

Slack et al. (Journal of Food Processing and Preservation, vol. 10, 1986, pages 19-30) explored a different approach and prepared BLG-enriched pellets by adjusting the pH using demineralized acid whey and sweet whey to a value of 4.65 and separating the resulting pellet by centrifugation and decantation. The resulting precipitate granules were described as being relatively insoluble and containing a significant amount of protein contaminants in additional BLG. No crystal formation was observed. It should be noted that the BLG precipitates that may form at pH 4.65 are not BLG crystals.

Palmer (Crystalline Globulin from Cow's Milk, J. Biol. Chem, Vol. 104, 1934, pages 359-372) reported a labor-intensive and time-consuming method for obtaining acid whey protein crystals by successively salting out unwanted proteins, adjusting pH, and dialysis to remove other unwanted proteins. Finally, upon receipt of a highly purified solution of BLG, crystallization of BLG occurred. The duration of the method was more than 12 days and required the addition of toluene. Therefore, the procedures disclosed in Palmer are inconsistent with safe food production and produce products that are unambiguously inedible.

Aschaffenburg et al. (Improved Method for the Preparation of Crystalline beta-Lactoglobulin and alphaLactalbumin from Cow's Milk, Bioch., vol. 65, 1957, pages 273-277) disclose an improved method over the Palmer method, the improvement providing beta-lactoglobulin crystals within a few days, not weeks. However, the improved method still requires the removal of unwanted proteins prior to crystallization and additionally uses toluene for crystallization, making it incompatible with safe food production.

JP H10218755 A discloses the preparation of cosmetic compositions containing a melanin production inhibitor which contains BLG as an active ingredient. In addition, this document shows that BLG, for example, can be isolated using the following process: hydrochloric acid is added to milk to precipitate casein, followed by filtration to obtain whey. The serum pH is adjusted to 6.0 and ammonium sulfate is added in an amount to half saturation; the precipitated protein is removed by salting out, and the filtrate is recovered. The filtrate is saturated with ammonium sulfate and the precipitated protein is recovered. The recovered protein is redissolved in water and subjected to dialysis at pH 5.2 to separate crystals, while β-lactoglobulin is obtained in a proportion equal to approximately 1.8 g per 1 liter of whey. However, the general steps of the proposed method described in JP H10218755 A do not lead to sufficient formation of BLG crystals. Thus, the document does not provide useful disclosure of BLG crystallization or BLG crystals.

US 2,790,790 discloses a process for precipitating proteins from solution, and more particularly the fractionated precipitation of relatively unconjugated proteins from an aqueous solution using sodium chloride as a precipitating agent. A method has been proposed for isolating BLG by NaCl mediated precipitation at pH 3.6-3.8. Example II of this document proposes that the precipitate obtained using NaCl can be dialyzed in the usual way to form crystalline B-lactoglobulin. However, US 2,790,790 does not show that the formation of BLG crystals at pH 3.6-3.8 is actually possible, and makes no reference to the value of a completely new method of dialysis of the BLG precipitate. Thus, the document does not provide useful disclosure of BLG crystallization or BLG crystals.

Brief description of the invention

The inventors made the surprising discovery that very high purity BLG can be obtained directly from a crude whey protein solution that contains significant amounts of other whey proteins besides BLG, and without the use of organic solvents such as toluene. This is contrary to the general knowledge of the prior art, which suggests that proteins must be largely purified before they can be crystallized, and that not all proteins can be crystallized.

This discovery has the potential to change the way whey protein is processed and fractionated in the dairy industry and makes available both efficient and gentle production of highly purified BLG that is safe to use as a food ingredient.

Thus, one aspect of the present invention relates to a method for preparing a food composition containing beta-lactoglobulin (BLG) in crystallized and/or isolated form, the method comprising the steps of:

a) providing a whey protein solution comprising BLG and at least one additional whey protein, said whey protein solution being supersaturated with BLG and having a pH in the range of 5-6;

b) crystallization of BLG in a supersaturated solution of milk whey protein; And

c) optionally separating the BLG crystals from the rest of the milk whey protein solution.

In addition, the present inventors have found that powdered whey protein food compositions that contain BLG crystals have significantly higher bulk densities than comparable prior art compositions. This is an advantage as it simplifies the handling of the powder and makes it less dusty.

Thus, another aspect of the present invention relates to a food composition containing beta-lactoglobulin in crystallized and/or isolated form, for example, obtained using one or more of the methods described herein. For example, the food composition may be a powder containing BLG crystals and having a bulk density of at least 0.40 g/mL. Alternatively, the food composition may be a liquid suspension or slurry containing BLG crystals.

In the context of the present invention, a dry product, such as, for example, a powder that contains BLG crystals, contains a product obtained by drying a suspension of BLG crystals, while the crystal structure of the wet BLG crystals could change during the drying process and, at least partially, could lose its characteristics determined with x-ray diffraction. Similarly, the terms dry BLG crystal and dried BLG crystal refer to a particle obtained by drying a wet BLG crystal, which dry particle does not necessarily itself have a crystalline structure. However, the present inventors have observed that when the dried BLG crystals are resuspended in cold (4°C) demineralized water at a weight ratio of 2 parts water to 1 part dried BLG crystals, the BLG crystal is rehydrated and acquires essentially the same crystal structure (type lattice symmetry and unit cell parameters), as before drying.

BLG is well known as an excellent source of essential amino acids, including, for example, leucine, so the BLG food composition provided by the present invention thus has several interesting uses in the food industry.

A further aspect of the present invention relates to an isolated BLG crystal having a rhombic space group P 21 21 21 and unit cell parameters a=68.68 (±5%) A, b=68.68 (±5%) A and c=156, 65 (±5%) A; and where the crystal has integral unit cell angles α=90°, β=90° and γ=90°.

Another aspect of the present invention relates to the use of a food composition as defined herein as a food ingredient.

A further aspect of the present invention relates to a food product containing a food composition as defined herein and a source of fat and/or a source of carbohydrates.

Brief description of graphic materials

In FIG. 1 shows two overlapping chromatograms of a crude whey protein solution (solid line) based on sweet whey and the resulting mother liquor after crystallization (dotted line). The difference between the solid and dotted lines is due to the removed BLG crystals.

fig. 2 is a microscopy photograph of the BLG crystals recovered in Example 1;

fig. 3 is a chromatogram of the recovered BLG crystal from Example 1;

fig. 4 is a plot of the conductivity of the milk whey protein solution versus the obtained yield of recovered BLG crystals;

fig. 5 is a graph of the temperature and conductivity of a whey protein solution versus the obtained yield of recovered BLG crystals;

in fig. 6 shows the relationship of total protein content (indicated indirectly by Brix degrees, which are proportional to protein content) in a milk whey protein solution and the resulting yield of recovered BLG crystals;

in fig. 7 shows chromatograms of raw material 1 from Example 3 (solid line) and mother liquor (dashed line) obtained after crystallization and removal of BLG crystals;

in fig. 8 is a microscopy photograph of a sample taken in the early stages of crystallization of raw material 1 from Example 3;

in fig. 9 is a microscopy photograph of a sample taken after the completion of crystallization of raw material 1 from Example 3;

in fig. 10 shows a chromatogram of washed BLG crystals obtained from raw material 1 of Example 3;

in fig. 11 shows chromatograms of raw material 2 from Example 3 (solid line) and mother liquor (dashed line) obtained after crystallization and removal of BLG crystals;

in fig. 12 is an image of the raw material 2 of Example 3 before (left image) and after (right image) crystallization;

in fig. 13 shows a microscopy photograph of BLG crystals, both whole and fragmented, obtained from raw material 2 of Example 3;

in fig. 14 and 15 show that an increase in conductivity or a change in the pH of the suspension of BLG crystals causes the BLG crystals to dissolve;

in fig. 17 shows raw material 3 from Example 3 before (left image) and after (right image) crystallization;

in fig. 18 is a microscopy photograph of BLG crystals recovered from raw material 3 of Example 3;

in fig. 19 shows a chromatogram of the recovered BLG crystal from raw material 3 of Example 3 without any washing step;

in fig. 20 shows the effect of increased conductivity on the yield of recovered BLG crystals;

in fig. 21 is a microscopy photograph of BLG crystals formed at a conductivity of 4.20 mS/cm;

in fig. 22 is a microscopy photograph of BLG crystals obtained in the early stages of crystallization of an SPC-based milk whey protein solution;

Fig. 23 shows the difference in bulk density of a standard whey protein isolate (WPI) and a highly purified BLG composition of the present invention, the composition containing BLG crystals;

in fig. 24 is a photograph of a centrifugal filter in which the BLG crystals from Example 3, Raw Material 1 were separated from the mother liquor;

in fig. 25 is a photograph of aliquots of six low phosphorus beverage samples of Example 8. From left to right, the aliquots represent samples A, B, C, D, E, and F;

in fig. 26 is a schematic representation of an embodiment of the crystallization process of Example 10 using DCF to separate the BLG crystals from the mother liquor;

in fig. 27 shows three photographs of the filter cake obtained during the separation of the BLG crystal and the mother liquor using a centrifuge filter.

Detailed description

As indicated above, one aspect of the present invention relates to a method for preparing a food composition containing beta-lactoglobulin (BLG) in crystallized and/or isolated form, the method comprising the steps of:

a) providing a whey protein solution comprising BLG and at least one additional whey protein, said whey protein solution being supersaturated with BLG and having a pH in the range of 5-6;

b) crystallization of BLG in a supersaturated solution of milk whey protein; And

c) optionally separating the BLG crystals from the rest of the milk whey protein solution.

In the context of the present invention, the term food composition refers to a composition that is safe for human consumption and used as a food ingredient and that does not contain problematic amounts of toxic ingredients such as toluene or other undesirable organic solvents. BLG is the most predominant protein in bovine and whey, and it exists in several genetic variants, with the main ones in bovine milk designated as A and B. BLG is a lipocalin protein and can bind many hydrophobic molecules, suggesting its role in their transport. BLG has additionally been shown to be able to bind iron via siderophores and may play a role in pathogen control. The BLG homologue is not found in human breast milk.

Bovine BLG is a relatively small protein of approx. 162 amino acid residues with a molecular weight of approx. 18.3-18.4 kDa. Under physiological conditions, the main

- 3 040134 it is dimeric, but dissociates into monomer at a pH below about 3, retaining its native state as determined by NMR. Conversely, BLG also exists in tetrameric, octameric, and other multimeric aggregation forms under various environmental conditions.

BLG solutions can gel under a variety of conditions when the native structure is sufficiently destabilized to allow aggregation. Prolonged heating at low pH and low ionic strength forms a transparent gel with thin filaments, in which protein molecules are assembled into long, rigid fibers.

In the context of the present invention, the term BLG or beta-lactoglobulin refers to BLG from different mammalian species, for example, in native and/or glycosylated forms, and it includes naturally occurring genetic variants.

In the context of the present invention, the term crystal refers to a solid material whose constituent components (such as atoms, molecules or ions) are arranged in a highly ordered microscopic structure, forming a crystal lattice that extends in all directions. BLG crystals are protein crystals that mainly contain BLG arranged in a highly ordered microscopic structure, forming a crystal lattice that extends in all directions. BLG crystals may, for example, be monolithic or polycrystalline, and may, for example, be intact crystals, fragments of crystals, or a combination thereof. Crystal fragments, for example, are formed if intact crystals are subjected to mechanical shear during processing. Crystal fragments also have a highly ordered microscopic crystal structure, but may not have a smooth surface and/or even edges or corners of an intact crystal. See, for example, FIG. 18 as an example of a number of intact BLG crystals and FIG. 13 as an example of BLG crystal fragments. Using optical microscopy, in both cases the BLG crystal or crystal fragments can be identified visually as distinctly structured, compact and interconnected structures. Often the BLG crystal or crystal fragments are at least partially transparent. In addition, protein crystals are known to be birefringent, and this optical property can be used to identify unknown particles that have a crystalline structure. Non-crystalline BLG aggregates, on the other hand, appear as loosely structured, opaque, and open or porous lumps of various sizes.

In the context of the present invention, the term crystallize refers to the formation of protein crystals. Crystallization can, for example, occur spontaneously or be initiated by the addition of crystallization seeds.

The food composition contains BLG in crystallized and/or isolated form. A food composition that contains BLG in an isolated form contains at least 80% wt./weight. BLG relative to total solids. A food composition that contains BLG in crystallized form contains at least some BLG crystals, but preferably a significant amount of BLG crystals.

BLG crystals can often be observed using microscopy, and they can even reach a size that makes them visible to the naked eye.

In the context of the present invention, a fluid that is supersaturated or supersaturated with BLG contains dissolved BLG at a concentration that is above the saturation point of BLG in that fluid under given physical and chemical conditions. The term supersaturated is well known in the field of crystallization (see, for example, Gerard Coquerela, Crystallization of molecular systems from solution: phase diagrams, supersaturation and other basic concepts, Chemical Society Reviews, p. 22862300, Issue 7, 2014), and supersaturation can be defined using a number of different measurement techniques (eg spectroscopy or particle size analysis). In the context of the present invention, BLG supersaturation is determined using the following procedure.

BLG liquid supersaturation test procedure under a specific set of conditions:

a) Dispense 50 ml of the sample fluid to be tested into a centrifuge tube (VWR part no. 525-0402) 115 mm high, 25 mm ID, 50 ml capacity. Be careful to keep the sample and its subsequent fractions in the original physical and chemical conditions of the liquid during steps a)-h);

b) immediately centrifuge the sample at 3000 g for 3.0 min to reach max acceleration in 30 s and max deceleration in 30 s;

c) immediately after centrifugation, transfer as much of the supernatant as possible (without shaking the pellets, if any) into a second centrifuge tube (same type as in step a);

d) remove a 0.05 ml aliquot of the supernatant (aliquot A);

e) add 10 mg of BLG crystals (at least 98% pure BLG based on total solids) with a particle size of 200 µm or less to a second centrifuge tube and shake the mixture;

f) allow the second centrifuge tube to stand for 60 minutes at the initial temperature

- 4 040134 ratute;

g) immediately after step f) centrifuge the second centrifuge tube at 500 g for 10 min and then remove another 0.05 ml aliquot of the supernatant (aliquot B);

h) remove the pellet from the centrifugation in step g), if present, resuspend it in milliQ water and check the suspension immediately for the presence of crystals that are visible by microscopy;

i) determine the concentration of BLG in aliquots A and B using the method described in example 9.9 results are expressed as % wt./wt. BLG relative to total aliquot weight. The concentration of BLG in aliquot A is called C _BLG , a, and the concentration of BLG in aliquot B is called C _BLG , c;

j) the liquid from which the sample of step a) was sampled was supersaturated (under specific conditions) if C _BLG , _B is lower than C _BLG , _A , and if crystals are observed in step i).

In the context of the present invention, the terms liquid and solution encompass compositions that contain a combination of liquid and solid or semi-solid particles, such as, for example, protein crystals or other protein particles. Therefore, the liquid or solution may be a suspension or even a slurry. However, the liquid and solution are preferably pumpable.

In some preferred embodiments of the present invention, the method does not include separation in step c) and provides a food composition that contains both BLG crystals and additional milk whey protein. In addition, if this method variant includes drying in step f), it provides a dry composition containing BLG crystals and additional milk whey protein, i.e. WPC or WPI wherein at least a portion of the BLG is present as BLG crystals. Preferably, the method comprises steps a), b) and f) in direct sequence.

If the raw material based on milk whey protein is milk whey protein concentrate (WPC), milk whey protein isolate (WPI), whey protein concentrate (SPC) or whey protein isolate (SPI), then the above method variant makes it possible to obtain WPC, WPI , SPC or SPI in liquid or dry form, in which at least a portion of the BLG is present in crystalline form.

The terms whey protein concentrate and whey protein concentration refer to dry or aqueous compositions in which the total amount of protein is 20-89% w/w. relative to total solids.

WPC or SPC preferably contains

20-89% w/w protein relative to total solids,

15-70% w/w BLG relative to total protein,

8-50% w/w ALA relative to total protein, and

0-40% w/w CMP for protein.

Alternatively, but preferably, the WPC or SPC may comprise

20-89% w/w protein relative to total solids,

15-90% w/w BLG relative to total protein,

4-50% w/w ALA relative to total protein; And

0-40% w/w CMP for protein.

Preferably WPC or SPC contains

20-89% w/w protein relative to total solids,

15-80% w/w BLG relative to total protein,

4-50% w/w ALA relative to total protein, and

0-40% w/w CMP for protein.

More preferably WPC or SPC contains

70-89% w/w protein relative to total solids,

30-90% w/w BLG relative to total protein,

4-35% w/w ALA relative to total protein, and

0-25% w/w CMP for protein.

The terms whey protein isolate and whey protein isolate refer to dry or aqueous compositions in which the total amount of protein is 90-100% w/w. relative to total solids.

WPI or SPI preferably contains

90-100% w/w protein relative to total solids,

15-70% w/w BLG relative to total protein,

8-50% w/w ALA relative to total protein, and

0-40% w/w CMP relative to total protein.

Alternatively, but preferably, the WPI or SPI may comprise

90-100% w/w protein relative to total solids,

30-95% w/w BLG relative to total protein,

4-35% w/w ALA relative to total protein, and

- 5 040134

0-25% w/w CMP relative to total protein.

More preferably the WPI or SPI may comprise

90-100% w/w protein relative to total solids,

30-90% w/w BLG relative to total protein,

4-35% w/w ALA relative to total protein, and

0-25% w/w CMP relative to total protein.

In some preferred embodiments of the present invention, the method further comprises step d) washing the BLG crystals, eg isolated BLG crystals obtained in step c).

In some preferred embodiments of the present invention, the method further comprises the step e) of recrystallizing the BLG crystals, for example the BLG crystals obtained in step c) or d).

The method may, for example, include stages a), b), c), d) and e) or even consist of them. Alternatively, the process may comprise or even consist of steps a), b), c) and e).

In some particularly preferred embodiments of the present invention, the method further comprises the step f) of drying the BLG-containing composition obtained in step b), c), d) or e).

The method may, for example, include stages a), b) and f) or even consist of them. Alternatively, the method may comprise or even consist of steps a), b), c) and f). Alternatively, the method may comprise or even consist of steps a), b), c), d) and f). Alternatively, the method may comprise or even consist of steps a), b), c), d), e) and f).

As said, step a) of the present invention comprises providing a milk whey protein solution that contains BLG and at least additional milk whey protein.

In the context of the present invention, the term milk whey protein refers to a protein that is present in milk whey. Milk whey protein from a solution of milk whey protein may be a subset of the different types of proteins present in milk whey, or it may be a complete set of different types of proteins present in milk whey. However, the milk whey protein solution always contains BLG.

In the context of the present invention, the term additional protein means a protein that is not a BLG. The additional protein that is present in the milk whey protein solution typically contains one or more non-BLG proteins that are present in milk whey. Non-limiting examples of such proteins are alpha-lactalbumin, bovine serum albumin, immunoglobulins, casein macropeptide (CMP), osteopontin, lactoferrin, and milk fat globule membrane proteins.

Thus, the milk whey protein solution may preferably contain at least one additional milk whey protein selected from the group consisting of alpha-lactalbumin, bovine serum albumin, immunoglobulins, casein macropeptide (CMP), osteopontin, lactoferrin, milk fat globule membrane proteins and their combinations.

Alpha-lactalbumin (ALA) is a protein present in the milk of almost all mammals. ALA forms the regulatory subunit of the lactose synthase (LS) heterodimer, and β-1,4 galactosyltransferase (beta4Gal-T1) forms the catalytic component. Together, these proteins provide the ability to produce lactose at the expense of LS by transferring galactose moieties to glucose. As a multimer, alpha-lactalbumin strongly binds calcium and zinc ions and may exhibit bactericidal or anticancer activity. One of the main structural differences with beta-lactoglobulin is that ALA does not have any free thiol group that can serve as a starting point for a covalent aggregation reaction. As a result, pure ALA will not gel upon denaturation and acidification.

In the context of the present invention, the term ALA or alpha-lactalbumin refers to alpha-lactalbumin of various mammalian species, eg in native and/or glycosylated forms, and includes naturally occurring genetic variants.

In some embodiments, the implementation of the present invention, the solution of whey protein contains no more than 10% wt./weight. casein relative to the total amount of protein, preferably not more than 5% wt./wt., more preferably not more than 1% wt./weight. and even more preferably not more than 0.5% casein relative to total protein. In some preferred embodiments of the present invention, the whey protein solution does not contain any detectable amount of casein.

The term whey refers to the liquid that remains after the removal of casein and milk fat globules from milk, for example by microfiltration or ultrafiltration through large pores. Whey may also be referred to as ideal whey.

The term whey protein or whey protein refers to the protein that is present in milk whey.

- 6 040134

The term whey refers to the liquid supernatant that remains after casein has been precipitated and removed from milk. Casein precipitation can, for example, be carried out by acidifying the milk and/or by means of rennet.

There are several types of whey, such as sweet whey, which is a whey product obtained by precipitation of casein based on the use of rennet, and sour whey, which is a whey product obtained by precipitation of casein based on the use of acid. Precipitation of casein based on the use of acid can, for example, be carried out by adding food acids or by means of bacterial cultures.

In some preferred embodiments of the present invention, the whey protein solution from stage a) contains at least 5% wt./weight. additional milk whey protein relative to total protein. Preferably, the whey protein solution from step a) contains at least 10% w/w. additional milk whey protein relative to total protein. More preferably, the whey protein solution from step a) contains at least 15% w/w. additional milk whey protein relative to total protein. Even more preferably, the whey protein solution from step a) contains at least 20% w/w. additional milk whey protein relative to total protein. Most preferably, the whey protein solution from step a) contains at least 30% w/w. additional milk whey protein relative to total protein.

In other preferred embodiments of the present invention, the whey protein solution from stage a) contains at least 1% wt./weight. additional milk whey protein relative to total protein. Preferably, the whey protein solution from step a) contains at least 2% w/w. additional milk whey protein relative to total protein. Even more preferably, the whey protein solution from step a) contains at least 3% w/w. additional milk whey protein relative to total protein. Most preferably, the whey protein solution from step a) contains at least 4% w/w. additional milk whey protein relative to total protein.

In still other preferred embodiments of the present invention, the whey protein solution from step a) contains at least 35% w/w. additional milk whey protein relative to total protein. Preferably, the whey protein solution from step a) contains at least 40% w/w. additional milk whey protein relative to total protein. More preferably, the whey protein solution from step a) may, for example, contain at least 45% w/w. additional milk whey protein relative to total protein. Even more preferably, the whey protein solution from step a) may contain at least 50% w/w. additional milk whey protein relative to total protein.

In some preferred embodiments of the present invention, the whey protein solution from stage a) contains in the range of 5-90% wt./weight. additional milk whey protein relative to total protein. Preferably, the whey protein solution from step a) may contain in the range of 10-80% w/w. additional milk whey protein relative to total protein. The solution of milk whey protein from stage a) may, for example, contain in the range of 20-70% wt./weight. additional milk whey protein relative to total protein. Preferably, the whey protein solution from stage a) contains in the range of 30-70% wt./weight. additional milk whey protein relative to total protein.

As already mentioned, the authors of the present invention found that the crystallization of BLG is possible without the use of organic solvents. This purification approach can also be used to purify milk whey protein formulations that have already undergone some BLG purification, and it provides simple methods that further increase the purity of BLG. Thus, in some preferred embodiments of the present invention, the whey protein solution from stage a) contains in the range of 1-20% wt./weight. additional milk whey protein relative to total protein. Preferably, the whey protein solution from stage a) may contain in the range of 2-15% wt./weight. additional milk whey protein relative to total protein. Even more preferably, the whey protein solution from stage a) may, for example, contain in the range of 3-10% wt./weight. additional milk whey protein relative to total protein.

In some embodiments, implementation of the present invention, the solution of whey protein from stage a) contains at least 5% wt./weight. ALA relative to total protein.

- 7 040134

Preferably, the whey protein solution from step a) contains at least 10% w/w. ALA relative to total protein. Even more preferably, the whey protein solution from step a) contains at least 15% w/w. ALA relative to total protein. Alternatively, the whey protein solution from step a) may contain at least 20% w/w. ALA relative to total protein.

In some preferred embodiments of the present invention, the whey protein solution from step a) contains at least 25% w/w. ALA relative to total protein. Preferably, the whey protein solution from step a) contains at least 30% w/w. ALA relative to total protein. The solution of milk whey protein from stage a) preferably contains at least 35% wt./weight. ALA relative to total protein. Even more preferably, the whey protein solution from step a) may contain at least 40% w/w. ALA relative to total protein.

In some preferred embodiments of the present invention, the whey protein solution from step a) contains ALA in the range of 5-95% w/w. relative to the total amount of protein. Preferably, the whey protein solution from step a) contains ALA in the range of 5-70% w/w. relative to the total amount of protein. Even more preferably, the whey protein solution from step a) may contain ALA in the range of 10-60% w/w. relative to the total amount of protein. The whey protein solution from step a) preferably contains ALA in the range of 12-50% w/w. relative to the total amount of protein. Even more preferably, the whey protein solution from step a) may contain ALA in the range of 20-45% w/w. relative to the total amount of protein.

In some preferred embodiments of the present invention, the whey protein solution from step a) has a weight ratio of BLG and ALA of at least 0.01. Preferably, the whey protein solution from step a) has a weight ratio of BLG and ALA of at least 0.5. Even more preferably, the milk whey protein solution from step a) has a weight ratio of BLG and ALA of at least 1, such as at least 2. For example, the milk whey protein solution from step a) can have a weight ratio of BLG and ALA of at least 3. All amounts and concentrations of BLG and other proteins in the milk whey protein solution and whey protein raw material are of dissolved protein and do not contain precipitated or crystallized protein.

In the context of the present invention, the term ratio by weight of component X and component Y means the value obtained by calculating m _X /m _Y , where m _X is the amount (weight) of the X components, and m _Y is the amount (weight) of the Y components.

In some preferred embodiments of the present invention, the whey protein solution from step a) has a weight ratio of BLG and ALA in the range of 0.01-20. Preferably the whey protein solution from step a) has a weight ratio of BLG and ALA in the range of 0.2-10. Even more preferably, the whey protein solution from step a) has a weight ratio of BLG and ALA in the range of 0.5-4. For example, the milk whey protein solution from step a) may have a weight ratio of BLG and ALA in the range of 1-3.

In some preferred embodiments of the present invention, the whey protein solution from stage a) contains at least 1% wt./weight. BLG relative to total protein. Preferably, the whey protein solution from step a) contains at least 2% w/w. BLG relative to total protein. Even more preferably, the whey protein solution from step a) contains at least 5% w/w. BLG relative to total protein. Preferably, the whey protein solution from step a) may contain at least 10% w/w. BLG relative to total protein.

In some preferred embodiments of the present invention, the whey protein solution from stage a) contains at least 12% wt./weight. BLG relative to total protein. For example, a solution of whey protein from stage a) may contain at least 15% wt./weight. BLG relative to total protein. The solution of milk whey protein from stage a) may contain at least 20% wt./weight. BLG relative to total protein. Alternatively, the whey protein solution from step a) may contain at least 30% w/w. BLG relative to total protein.

In some particularly preferred embodiments of the present invention, the whey protein solution from stage a) contains no more than 95% wt./weight. BLG relative to total protein. Preferably, the whey protein solution from step a) may contain no more than 90% w/w. BLG relative to total protein. More preferably, the whey protein solution from stage a) may, for example, contain no more than 85% wt./weight. BLG relative to total protein. Even more preferably, the whey protein solution from stage a) may, for example, contain no more than 80% wt./weight. BLG relative to total

- 8 040134 proteins. Preferably, the whey protein solution from step a) may contain no more than

78% w/w BLG relative to total protein. Preferably, the whey protein solution from step a) may contain no more than 75% w/w. BLG relative to total protein.

In some preferred embodiments of the present invention, the whey protein solution from step a) contains BLG in the range of 1-95% w/w. relative to the total amount of protein. Preferably, the whey protein solution from step a) may contain BLG in the range of 5-90% w/w. relative to the total amount of protein. More preferably, the whey protein solution from step a) contains BLG in the range of 10-85% w/w. relative to the total amount of protein. Even more preferably, the whey protein solution from step a) contains BLG in the range of 10-80% w/w. relative to the total amount of protein. Most preferably, the whey protein solution from step a) may contain BLG in the range of 20-70% w/w. relative to the total amount of protein.

In other preferred embodiments of the present invention, the whey protein solution from step a) contains BLG in the range of 10-95% w/w. relative to the total amount of protein. Preferably, the whey protein solution from step a) may contain BLG in the range of 12-90% w/w. relative to the total amount of protein. More preferably, the whey protein solution from step a) contains BLG in the range of 15-85% w/w. relative to the total amount of protein. Even more preferably, the whey protein solution from step a) contains BLG in the range of 15-80% w/w. relative to the total amount of protein. Most preferably, the whey protein solution from step a) may contain BLG in the range of 30-70% w/w. relative to the total amount of protein.

In some preferred embodiments of the present invention, the whey protein solution from step a) contains at least 0.4% w/w. BLG relative to the weight of the milk whey protein solution. Preferably, the whey protein solution contains at least 1.0% w/w. BLG. More preferably, the whey protein solution contains at least 2.0% w/w. BLG. Even more preferably, when the solution of whey protein contains at least 4% wt./weight. BLG.

Higher concentrations of BLG are even more preferred, and preferably the whey protein solution contains at least 6% w/w. BLG. More preferably, the whey protein solution contains at least 10% w/w. BLG. Even more preferably, when the solution of whey protein contains at least 15% wt./weight. BLG.

In some preferred embodiments of the present invention, the solution of whey protein from stage a) contains in the range of 0.4-40% wt./weight. BLG relative to the weight of the whey protein solution. Preferably, the whey protein solution contains BLG in the range of 1-35% w/w. More preferably, the whey protein solution contains BLG in the range of 4-30% w/w. Even more preferably, the whey protein solution contains BLG in the range of 10-25% w/w.

Any suitable source of milk whey protein can be used to prepare a solution of milk whey protein. In some preferred embodiments of the present invention, the milk whey protein solution comprises or even consists of whey protein concentrate, milk whey protein concentrate, whey protein isolate, milk whey protein isolate, or combinations thereof.

Preferably, the whey protein solution is a demineralized whey protein solution.

In this context, the term demineralized means that the conductivity of the milk whey protein solution is not more than 15 mS/cm, and preferably not more than 10 mS/cm, and even more preferably not more than 8 mS/cm. The ultrafiltrate (UF) conductivity of the demineralized whey protein solution is preferably at most 7 mS/cm, more preferably at most 4 mS/cm, and even more preferably at most 1 mS/cm.

It is particularly preferred that the milk whey protein solution is a demineralized whey protein concentrate, a demineralized whey protein isolate, a demineralized whey protein concentrate or a demineralized whey protein isolate.

In some particularly preferred embodiments of the present invention, the whey protein solution comprises or even consists of demineralized and pH-adjusted whey protein concentrate, whey protein concentrate, whey protein isolate, milk whey protein isolate, or combinations thereof.

The milk whey protein solution may, for example, contain or even consist of a demineralized whey protein concentrate. Alternatively, the milk whey protein solution may contain demineralized milk whey protein concentrate or

- 9 040134 even consist of it. Alternatively, the milk whey protein solution may contain or even consist of demineralized whey protein isolate. Alternatively, the milk whey protein solution may contain or even consist of demineralized milk whey protein isolate.

In the context of the present invention, the terms milk whey protein concentrate and whey protein concentrate refer to preparations based on whey or milk whey, which contain a protein in the range of approx. 20-89% w/w relative to total solids.

In the context of the present invention, the terms whey protein isolate and whey protein isolate refer to preparations based on whey or milk whey, which contain at least 90% wt./weight. protein relative to total solids.

The terms consists essentially of and consisting essentially of mean that the feature or characteristic of interest encompasses certain materials or steps and those that do not physically affect the essential and novel features of the claimed invention.

The protein from the milk whey protein solution is preferably obtained from the milk of mammals, and preferably from the milk of ruminants such as, for example, cow, sheep, goat, buffalo, camel, llama, mare and/or deer. A protein derived from bovine (cow) milk is particularly preferred. The BLG and additional milk whey protein are therefore preferably bovine BLG and bovine milk whey protein.

The protein from the milk whey protein solution is preferably as close to its native state as possible and preferably has been subjected to only moderate heat treatments or not at all.

In some preferred embodiments of the present invention, the BLG from the milk whey protein solution has a lactosylation degree of at most 1. Preferably, the BLG from the milk whey protein solution has a lactosylation degree of at most 0.6. More preferably, the BLG from the whey protein solution has a lactosylation degree of at most 0.4. Even more preferably, the BLG from the whey protein solution has a lactosylation degree of at most 0.2. Most preferably, the BLG from the whey protein solution has a lactosylation degree of at most 0.1, such as preferably at most 0.01.

The degree of lactosylation of BLG is determined according to Czerwenka et al. (J. Agric. Food Chem., Vol. 54, No. 23, 2006, pages 8874-8882).

In some preferred embodiments of the present invention, the whey protein solution has a furosin content of no more than 80 mg/100 g of protein. Preferably, the milk whey protein solution has a furosin content of at most 40 mg/100 g of protein. More preferably, the milk whey protein solution has a furosin content of at most 20 mg/100 g of protein. Even more preferably, the milk whey protein solution has a furosin content of at most 10 mg/100 g of protein. Most preferably, the milk whey protein solution has a furosin content of at most 5 mg/100 g of protein, such as preferably a furosin content of 0 mg/100 g of protein.

The milk whey protein solution usually contains other components besides the protein. The milk whey protein solution may contain other components that are normally present in whey or milk whey, such as minerals, carbohydrates and/or lipids. Alternatively or additionally, the milk whey protein solution may contain components that are not found in whey or milk whey. However, such non-intrinsic components should preferably be safe for use in food production and preferably also for human consumption.

This method is particularly preferred for isolating BLG from crude whey protein solutions that contain other solids other than BLG.

The milk whey protein solution may, for example, contain carbohydrates such as lactose, oligosaccharides and/or lactose hydrolysis products (ie glucose and galactose). The whey protein solution may, for example, contain carbohydrates in the range of 0-40% w/w, such as in the range of 1-30% w/w. or in the range of 2-20% wt./weight.

In some preferred embodiments of the present invention, the whey protein solution contains no more than 20% wt./weight. carbohydrates, preferably not more than 10% wt./weight. carbohydrates, more preferably not more than 5% wt./weight. carbohydrates and even more preferably not more than 2% wt./weight. carbohydrates.

The milk whey protein solution may also contain lipids, for example in the form of triglycerides and/or other types of lipids such as phospholipids.

- 10 040134

In some embodiments, implementation of the present invention in a solution of whey protein from stage a) the total amount of lipids is not more than 15% wt./weight. relative to total solids. Preferably, in the whey protein solution from step a), the total amount of lipids is not more than 10% w/w. relative to total solids. More preferably, in the whey protein solution from step a), the total lipid content is not more than 6% w/w. relative to total solids. Even more preferably, in the whey protein solution from step a), the total lipid content is not more than 1.0% w/w. relative to total solids. Most preferably, in the whey protein solution from step a), the total lipid content is not more than 0.5% w/w. relative to total solids.

The total amount of protein in a solution of whey protein is usually at least 1% wt./weight. relative to the weight of the milk whey protein solution. Preferably, the total amount of protein in the whey protein solution is at least 5% w/w. More preferably, the total amount of protein in the whey protein solution is at least 10% w/w. Even more preferably, the total amount of protein in the whey protein solution is at least 15% w/w.

In some preferred embodiments of the present invention, the total amount of protein in the whey protein solution is in the range of 1-50% w/w. Preferably, the total amount of protein in the whey protein solution is in the range of 540% w/w. More preferably, the total amount of protein in the whey protein solution is in the range of 10-30% w/w. Even more preferably, the total amount of protein in the whey protein solution is in the range of 15-25% w/w.

The total amount of protein in the milk whey protein solution is determined according to example 9.2.

The milk whey protein solution is typically prepared by adjusting one or more properties of the milk whey protein raw material, resulting in a milk whey protein solution that is supersaturated in BLG.

The raw material is preferably WPC, WPI, SPC, SPI, or a combination thereof.

In the context of the present invention, the term milk whey protein raw material refers to a composition that is converted into a solution of milk whey protein supersaturated with BLG. The raw material based on milk whey protein is usually an aqueous liquid containing BLG and at least one additional milk whey protein, but usually it is not supersaturated in BLG.

Embodiments relating to the chemistry of the milk whey protein solution also apply to the milk whey protein raw material, however, typically at least one parameter of the milk whey protein raw material is adjusted to avoid supersaturation or at least spontaneous crystallization.

In some preferred embodiments of the present invention, a supersaturated whey protein solution is obtained by making one or more of the following adjustments to the whey protein raw material:

pH adjustments;

decrease in conductivity;

lowering the temperature;

increase in protein concentration;

adding an agent that reduces water activity;

modification of the ionic composition.

In some preferred embodiments of the present invention, obtaining a solution of milk whey protein includes adjusting the pH of the raw material based on milk whey protein to a pH in the range of 5-6.

All pH values are measured with a pH glass electrode and normalized at 25°C.

The milk whey protein solution may, for example, have a pH in the range of 4.9-6.1. For example, the pH of a whey protein solution may be in the range of 5.0-6.1. Alternatively, the pH of the milk whey protein solution may be in the range of 5.1-6.1. Preferably the pH of the whey protein solution is in the range of 5.1-6.0.

In some preferred embodiments of the present invention, the pH of the whey protein solution is in the range of 5.0-6.0. Preferably the pH of the whey protein solution is in the range of 5.1-6.0. More preferably, the pH of the milk whey protein solution is in the range of 5.1-5.9. Even more preferably, the pH of the whey protein solution may be in the range of 5.2-5.9. Most preferably, the pH of the milk whey protein solution is in the range of 5.2-5.8.

- 11 040134

The pH is preferably adjusted with food acids and/or bases. Particular preference is given to food acids such as, for example, carboxylic acids. Suitable examples of such acids are, for example, hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, maleic acid, tartaric acid, lactic acid, citric acid or gluconic acid and/or mixtures thereof.

In some preferred embodiments of the present invention, the pH is adjusted using a lactone, such as, for example, D-glucono-delta-lactone, which slowly hydrolyzes and simultaneously lowers the pH of the aqueous liquid containing it. The target pH after lactone hydrolysis is complete can be accurately determined.

Useful examples of acceptable food bases, for example, are hydroxide sources such as, for example, sodium hydroxide, potassium hydroxide, calcium hydroxide, food acid salts, such as, for example, trisodium citrate, and/or combinations thereof.

In other preferred embodiments of the present invention, the pH is adjusted by adding a cation exchange material in its H+ form. The granular/large particle size cation exchange material is easily removed from the whey protein solution before crystallization or even after crystallization. Adjusting the pH by adding the cation exchange material in its H+ form is particularly advantageous in the present invention because it lowers the pH without adding negative counterions that significantly affect the conductivity of the milk whey protein raw material.

In some preferred embodiments of the present invention, obtaining a solution of milk whey protein includes reducing the conductivity of the raw material based on whey protein.

The conductivity values given in this document have been normalized at 25°C unless otherwise noted.

The authors found that reducing the conductivity of the milk whey protein solution resulted in a higher yield of BLG crystals. The minimum achievable conductivity of a whey protein solution depends on the composition of the protein fraction and the lipid fraction (if present). Some types of proteins, such as, for example, casein macropeptide (CMP), contribute more to conductivity than other types of proteins. Thus, it is preferable that the conductivity of the raw material based on milk whey protein is brought almost to the level at which protein and protein counterions are the main components contributing to the conductivity.

Conductivity reduction often involves the removal of at least some of the small free ions that are present in the liquid phase and are loosely bound to proteins.

It is often preferred that the milk whey protein solution has a conductivity of no more than 10 mS/cm. In some preferred embodiments of the present invention, the whey protein solution has a conductivity of at most 5 mS/cm. Preferably, the milk whey protein solution has a conductivity of at most 4 mS/cm.

Lower conductivities are even more preferred and result in higher yields of BLG crystals. Thus, the milk whey protein solution preferably has a conductivity of at most 3 mS/cm. In some preferred embodiments of the present invention, the whey protein solution has a conductivity of at most 1 mS/cm. Preferably, the milk whey protein solution has a conductivity of at most 0.5 mS/cm.

The conductivity of the raw material based on milk whey protein is preferably reduced by dialysis or diafiltration. Diafiltration by ultrafiltration is particularly preferred because it allows salts and small charged molecules to be washed out while proteins are preserved. In some preferred embodiments of the present invention, a single ultrafiltration unit is used for ultrafiltration/diafiltration and subsequent concentration of raw material based on whey protein.

The present inventors have found indications that the ratio of conductivity (expressed in mS/cm) and total protein in the milk whey protein solution (expressed as wt% of total protein relative to the total weight of milk whey protein solution) can preferably be maintained at or below a certain threshold value in order to facilitate the crystallization of BLG.

In some preferred embodiments of the present invention, the ratio of conductivity to total protein in the whey protein solution is not more than 0.3. Preferably, the ratio of conductivity to total protein in the milk whey protein solution is not more than 0.25. Preferably, the ratio of conductivity to total protein in the milk whey protein solution is not more than 0.20. More preferably, the ratio of conductivity to total protein in the milk whey protein solution is not more than 0.18. Even more preferably, the ratio of conductivity to total protein in the milk whey protein solution is not more than 0.12. Most preferably, the ratio of conductivity to total protein in the milk whey protein solution is not more than 0.10.

Preferably, for example, when the ratio of conductivity to total protein in the whey protein solution is approx. 0.07 or even lower.

In addition, the present inventors have found that the milk whey protein raw material can preferably be processed to provide a milk whey protein solution with an ultrafiltrate conductivity of not more than 10 mS/cm. The conductivity of the ultrafiltrate is a measure of the conductivity of the small molecule fraction of a liquid and is measured according to Example 9.10. As the term conductivity itself is used in this document, it refers to the conductivity of the fluid of interest. If the term ultrafiltrate conductivity is used, it refers to the conductivity of the low molecular weight fraction of the liquid and is measured according to example 9.10.

Preferably, the conductivity of the ultrafiltrate of the milk whey protein solution is not more than 7 mS/cm. More preferably, the conductivity of the ultrafiltrate of the milk whey protein solution may be no more than 5 mS/cm. Even more preferably, the conductivity of the ultrafiltrate of the milk whey protein solution may be no more than 3 mS/cm.

Even lower ultrafiltrate conductivities can be used, which are particularly preferred if a high BLG yield is to be obtained. Thus, preferably, the conductivity of the ultrafiltrate of the milk whey protein solution is not more than 1.0 mS/cm. More preferably, the conductivity of the ultrafiltrate of the milk whey protein solution may be no more than 0.4 mS/cm. Even more preferably, the conductivity of the ultrafiltrate of the milk whey protein solution may be no more than 0.1 mS/cm. Most preferably, the conductivity of the ultrafiltrate of the milk whey protein solution may be no more than 0.04 mS/cm.

Even lower ultrafiltrate conductivity values can be achieved, eg MilliQ water is used as a diafiltration diluent (MilliQ water has a conductivity of approx. 0.06 µS/cm). Thus, the conductivity of the ultrafiltrate solution of milk whey protein can be no more than 0.01 mS/cm. Alternatively, the conductivity of the ultrafiltrate of the milk whey protein solution may be no more than 0.001 mS/cm. Alternatively, the conductivity of the ultrafiltrate of the milk whey protein solution may be no more than 0.0001 mS/cm.

In some preferred embodiments of the present invention, obtaining a solution of milk whey protein includes reducing the temperature of the raw material based on milk whey protein.

For example, preparing a whey protein solution may include lowering the temperature of the whey protein raw material to at least 5°C, preferably at least 10°C, and even more preferably at least 15°C. For example, obtaining a solution of milk whey protein may include reducing the temperature of the raw material based on whey protein to at least 20°C.

The temperature of the raw material based on whey protein, for example, can be reduced to not more than 30°C, preferably not more than 20°C, and even more preferably to not more than 10°C. The inventors have found that even lower temperatures provide a higher degree of supersaturation, so that the temperature of the milk whey protein raw material can, for example, be reduced to no more than 5°C, preferably no more than 2°C, and even more preferably no more than 0 °C. The temperature may even be below 0° C., however, preferably the whey protein solution should remain in a pumpable state, for example as an ice slurry.

In some preferred embodiments of the present invention, the whey protein solution is an ice slurry prior to initiating BLG crystallization. Alternatively or additionally, the whey protein crystallizing solution may be converted to or maintained as an ice slurry during the BLG crystallization in step b).

In some particularly preferred embodiments of the present invention, obtaining a solution of milk whey protein includes increasing the concentration of total protein in the raw material based on milk whey protein. The milk whey protein raw material, for example, may be subjected to one or more protein concentration steps such as ultrafiltration, nanofiltration, reverse osmosis and/or evaporation, thereby concentrating to form a milk whey protein solution.

Ultrafiltration is particularly preferred because it allows selective protein concentration while salt and carbohydrate concentrations remain virtually unchanged. As stated above, ultrafiltration is preferably used for both diafiltration and concentration of the milk whey protein raw material.

In some preferred embodiments of the present invention, the concentration of BLG in the whey protein solution is below the level at which spontaneous crystallization of BLG occurs. Thus, it is often preferable to stop the fortification of the whey protein raw material when the milk whey protein solution is in the meta-stable region, i.e. in the supersaturated region, in which BLG crystals can grow when seeded, but in which crystallization does not start spontaneously.

In some preferred embodiments of the present invention, the preparation of the milk whey protein solution comprises adding one or more water reducing agents to the milk whey protein raw material.

Applicable, but not limiting, examples of such water activity reducing agents are polysaccharides and/or polyethylene glycol (PEG).

In some preferred embodiments of the present invention, the preparation of the milk whey protein solution comprises modifying the ionic composition of the milk whey protein raw material, for example, by ion exchange, addition of new ions, dialysis, or diafiltration.

Typically, a whey protein solution is prepared by combining two or more of the above process steps to provide supersaturation.

In some preferred embodiments of the present invention, obtaining a solution of milk whey protein includes exposing the raw material based on milk whey protein with at least the following:

concentration, for example, using ultrafiltration, nanofiltration or reverse osmosis at temperatures above 10°C; and subsequent cooling to a temperature below 10°C.

In other preferred embodiments of the present invention, the preparation of the milk whey protein solution comprises exposing the milk whey protein raw material to at least the following:

concentration at a pH above 6.0; and then lowering the pH by adding an acid (eg GDL or cation exchange material in the form of H ⁺ ).

In still other preferred embodiments of the present invention, the preparation of the milk whey protein solution comprises applying to the milk whey protein raw material with at least the following:

decrease in conductivity, for example, by diafiltration using a membrane that retains at least BLG.

In additional preferred embodiments of the present invention, obtaining a milk whey protein solution comprises exposing a raw material based on milk whey protein with a combination of at least the following:

bringing the pH to 5-6;

reducing conductivity by diafiltration using a membrane that retains at least BLG;

protein concentration, for example, using ultrafiltration, nanofiltration or reverse osmosis at temperatures above 10°C; and finally, cooling to a temperature below 10°C.

In addition, the authors of the present invention found that the yield of BLG in the method of the present invention can be improved by controlling the molar ratio of the total amount of sodium + potassium and the total amount of calcium and magnesium. A higher relative amount of calcium and magnesium obviously means an increase in the yield of BLG, and thus an increase in the efficiency of extraction of BLG in the method of the present invention.

In some preferred embodiments of the present invention, the whey protein solution from step a) has a Na+K to Ca+Mg molar ratio of not more than 4. More preferably, the whey protein solution from step a) has a Na+K to Ca+Mg molar ratio of not more than more than 2. Even more preferably, the whey protein solution from step a) has a Na+K to Ca+Mg molar ratio of at most 1.5 and even more preferably at most 1.0. Most preferably, the whey protein solution from step a) has a Na+K to Ca+Mg molar ratio of at most 0.5, such as at most 0.2.

The molar ratio of Na+K and Ca+Mg is calculated as (m _Na +m _K )/(m _Ca +m _Mg ), where m _Na is the content of elemental Na in moles, m _K is the content of elemental K in moles, m _Ca is the content elemental Ca in moles, am _Mg represents the content of elemental Mg in moles.

- 14 040134

It is particularly preferred that the whey protein solution is supersaturated with

BLG by salting in and so that the BLG can be crystallized from the whey protein solution in the salting-in mode.

In some embodiments of the present invention, the whey protein solution is characterized by a low content of denatured protein, especially if the BLG-based food product of the present invention is also to be characterized by a certain degree of protein denaturation. Preferably the whey protein solution has a protein denaturation of at most 2%, preferably at most 1.5%, more preferably at most 1.0%, and most preferably at most 0.8%.

Step b) of the method comprises crystallizing at least some of the BLG in a supersaturated milk whey protein solution.

It is especially preferred that the crystallization in step b) takes place in the salting-in mode, i.e. in a liquid that is characterized by low ionic strength and conductivity. This is different from the salting out mode, in which significant amounts of salts are added to the solution to promote crystallization.

Crystallization of BLG in step b) may, for example, include one or more of the following: waiting for crystallization to proceed;

adding seeds for crystallization;

an even greater increase in the values of the degree of supersaturation according to BLG; and/or mechanical impact.

In some preferred embodiments of the present invention, step b) comprises adding a crystallization seed to the milk whey protein solution. The authors have found that the addition of a crystallization seed provides the ability to control when and where the BLG crystallization will take place to avoid sudden plugging of process equipment and unanticipated stoppages during production. For example, it is often advantageous to avoid the onset of crystallization while concentrating the raw material based on milk whey protein.

In principle, any seed material that initiates BLG crystallization can be used. However, it is preferred that hydrated BLG crystals or dried BLG crystals be used for seeding to avoid adding additional impurities to the milk whey protein solution.

The crystallization seeds may be in dry form or may form part of a suspension when added to the whey protein solution. The addition of a slurry containing crystallization seeds, such as BLG crystals, is preferred in the present invention because it has been found to provide a faster onset of crystallization. Preferably, such a suspension containing a crystallization seed has a pH in the range of 5-6 and a conductivity of not more than 10 mS/cm.

In some embodiments, implementation of the present invention, at least part of the seeds for crystallization is in the solid phase, which comes into contact with a solution of milk whey protein.

The crystallization seeds preferably have a smaller particle size than the BLG crystals of the desired size. The size of the seeds for crystallization can be changed by removing the largest seed particles using sieving or other size fractionation methods. Particle size reduction, for example by milling, can also be carried out prior to particle size fractionation.

In some embodiments of the present invention, at least 90% wt./weight. seeds for crystallization are characterized by a particle size (measured by sieve analysis) in the range of 0.1-600 microns. For example, at least 90% wt./weight. seeds for crystallization can be characterized by a particle size in the range of 1-400 microns. Preferably at least 90% wt./weight. seeds for crystallization can be characterized by a particle size in the range of 5-200 microns. More preferably at least 90% wt./weight. seeds for crystallization can be characterized by a particle size in the range of 5-100 microns.

Particle size and dose of crystallization seeds can be optimized to ensure optimal crystallization of BLG.

In some preferred embodiments of the present invention, crystallization seeds are added to the whey protein raw material prior to reaching BLG supersaturation, but preferably in such a way that at least some of the crystallization seeds is still present when supersaturation is reached. For example, this can be done by adding a crystallization seed when the whey protein raw material is close to supersaturated, such as during cooling, concentration and/or pH adjustment, and to achieve supersaturation before completely dissolving the crystallization seed.

- 15 040134

In some preferred embodiments of the present invention, step b) comprises further increasing the degree of BLG supersaturation, preferably to the extent that the BLG crystallizes immediately, ie. no more than 20 minutes, and preferably no more than 5 minutes. This is also called the nucleation zone, where crystallites spontaneously form and the crystallization process begins.

For example, the degree of supersaturation can be increased by using one or more of the following:

an even greater increase in the concentration of protein in the solution of milk whey protein, an even greater cooling of the solution of milk whey protein;

bringing the solution of milk whey protein to a pH value close to optimal for BLG crystallization;

further reduction in conductivity.

In some preferred embodiments of the present invention, step b) includes waiting for the formation of BLG crystals. This can take several hours and is typical of a whey protein solution that is only slightly supersaturated with BLG and has not been seeded for crystallization.

In some preferred embodiments of the present invention, it is stipulated that the provision of the whey protein solution (step a) and the crystallization of the BLG (step b) occur in two separate steps.

However, in other preferred embodiments of the present invention, step b) comprises further adjusting the crystallization of the milk whey protein solution to increase the degree of BLG supersaturation, or at least maintain supersaturation. Additional adjustment leads to an increase in the yield of BLG crystals.

Such additional adjustment may include one or more of further increasing the protein concentration in the crystallizing milk whey protein solution;

cooling the crystallizing whey protein solution to an even lower temperature;

bringing the crystallizing whey protein solution even closer to the optimum pH for BLG crystallization;

an even greater decrease in the conductivity of the crystallizing solution of milk whey protein.

In some preferred embodiments of the present invention, the crystallizing milk whey protein solution is maintained in the metastable zone during step b) to avoid spontaneous formation of new crystallites.

The authors determined the crystal lattice structure of the isolated BLG crystals by X-ray crystallography and did not find a similar crystal in the prior art.

In some preferred embodiments of the present invention, at least some of the BLG crystals obtained during step b) have an orthorhombic space group P 2 ₁ 2 ₁ 21.

Preferably, at least some of the resulting BLG crystals have a rhombic space group P 21 21 21 and unit cell parameters a=68.68 (±5%) A, b=68.68 (±5%) A and c=156, 65 (±5%) A; and integral unit cell angles α=90°, β=90° and γ=90°.

In some preferred embodiments of the present invention, at least some of the resulting BLG crystals have a rhombic space group P 21 21 21 and unit cell parameters a=68.68 (±2%) A, b=68.68 (±2% ) A and c=156.65 (±2%) A; and integral unit cell angles α=90°, β=90° and γ=90°.

Even more preferably, at least some of the resulting BLG crystals may have a rhombic space group P 21 21 21 and unit cell parameters a=68.68 (±1%) A, b=68.68 (±1%) A and c =156.65 (±1%) A; and integral unit cell angles α=90°, β=90° and γ=90°.

Most preferably, at least some of the resulting BLG crystals have a rhombic space group P 21 21 21 and unit cell parameters a=68.68 A, b=68.68 A and c=156.65 A; and integral unit cell angles α=90°, β=90° and γ=90°.

In some particularly preferred embodiments of the present invention, the method comprises step c) separating at least some of the BLG crystals from the remaining whey protein solution. This is particularly preferred when BLG purification is required.

Stage c) may, for example, provide for the separation of BLG crystals to achieve a solids content of at least 30% wt./weight. Preferably, step c) involves separating BLG crystals to achieve a solids content of

- 16 040134 at least 40% w/w Even more preferably, step c) involves separating BLG crystals to achieve a solids content of at least 50% w/w.

The authors found that a high solids content is preferable for BLG purification, since the aqueous part that adheres to the isolated BLG crystals usually contains impurities that should not be tolerated. Additionally, the high solids content reduces the energy required to convert the separated BLG crystals into a dry product such as powder, and it increases the yield of BLG produced in the dryer at a given capacity.

In some preferred embodiments of the present invention, step c) involves separating BLG crystals to achieve a solids content of at least 60%. Preferably, step c) involves separating the BLG crystals to achieve a solids content of at least 70%. Even more preferably, step c) involves separating the BLG crystals to achieve a solids content of at least 80%.

In some preferred embodiments of the present invention, the separation in step c) comprises one or more of the following process steps:

centrifugation;

decantation;

filtration;

precipitation;

combinations of the above.

These typical technological operations are well known to a person skilled in the art and are easily carried out. Separation by filtration may, for example, involve the use of vacuum filtration, dynamic tangential flow filtration (DCF), filter presses or filter centrifuges.

Different pore sizes for filtration can be used based on the desired output. Preferably, the filter allows native whey protein and small aggregates to pass, but retains BLG crystals. The filter preferably has a nominal pore size of at least 0.1 microns. The filter may, for example, have a nominal pore size of at least 0.5 µm. Even more preferably, the filter may have a nominal pore size of at least 2 microns.

Filters with large pore sizes can also be used and are in fact preferred if mainly large crystals are to be separated from a liquid containing BLG crystals. In some embodiments of the present invention, the filter has a nominal pore size of at least 5 microns. Preferably the filter has a nominal pore size of at least 20 microns. Even more preferably, the filter may have a pore size of at least 40 microns.

The filter may, for example, have a pore size in the range of 0.03-5000 µm, such as 0.1-5000 µm. Preferably, the filter may have a pore size in the range of 0.5-1000 microns. Even more preferably, the filter may have a pore size in the range of 5-800 µm, such as in the range of 10-500 µm or in the range of 50-500 µm.

In some preferred embodiments of the present invention, the filter has a pore size in the range of 0.03-100 microns. Preferably, the filter may have a pore size in the range of 0.150 µm. More preferably, the filter may have a pore size in the range of 4-40 microns. Even more preferably, the filter may have a pore size in the range of 5-30 µm, such as in the range of 10-20 µm.

The advantage of using filters with a pore size greater than 1 μm is that bacteria and other microorganisms are also at least partially removed during separation and also optionally during washing and/or recrystallization. Thus, the method of the present invention makes it possible to produce highly purified BLG at a very low bacterial load, thereby preventing thermal damage to the protein.

Another advantage of using filters with a pore size greater than 1 µm is that the removal of water and subsequent drying becomes easier and less energy intensive.

The remaining milk whey protein solution that is separated from the BLG crystals can be recycled to the milk whey protein raw material during the preparation of the milk whey protein solution.

In some preferred embodiments of the present invention, a centrifuge filter is used in step c). In other preferred embodiments of the present invention, a decanter centrifuge is used in step c). The initial results (see example 13) showed that the use of a centrifuge filter and/or a decanter centrifuge to separate the BLG crystals from the mother liquor provides more reliable operation of the process than, for example, vacuum filtration.

It is often preferable to dry the resulting filter cake.

- 17 040134 with a drying gas in order to reduce the moisture content of the filter cake, and preferably to enable separation of the filter cake from the filter. The use of a drying gas may form part of the separation step or alternatively the final drying step if the filter cake is converted directly into a dry food composition based on

BLG.

In some preferred embodiments of the present invention, a DCF unit is used in step c).

Initial tests (see example 12) showed that the use of a DCF unit with a membrane pore size in the range of 0.03-5 µm, and preferably in the range of 0.3-1.0 µm, provides effective separation of BLG crystals, and the authors observed that The DCF plant can operate for a period of time sufficient to separate crystals from even large batches of whey protein solution containing BLG crystals.

In some preferred embodiments of the present invention, step c) is performed with a DCF plant equipped with a membrane capable of retaining BLG crystals, wherein the DCF filtrate is recycled to form a portion of the whey protein solution or milk whey protein raw material, and the retentate DCF can be recovered or returned to the crystallization tank. Preferably, the DCF filtrate is treated, for example, by ultra-/diafiltration, to make it supersaturated with BLG before mixing with the milk whey protein solution or milk whey protein raw material.

Preferably, these embodiments do not require a rise in temperature of the liquid streams above 15° C., so they are less prone to microbial contamination than process embodiments that require higher temperatures. Another industrial advantage of these embodiments is that the level of supersaturation is easily controlled and can be maintained at a level where spontaneous crystallization is undesirable. Thus, the temperature of the liquid streams in these process embodiments is preferably at most 15°C, more preferably at most 12°C, and even more preferably at most 10°C, and most preferably at most 5°C.

These embodiments are exemplary in Example 10 and shown in FIG. 26. These embodiments can be carried out either as a batch process or as a continuous process.

In some preferred embodiments of the present invention, the method comprises step d) washing the BLG crystals, eg the BLG crystals separated in step c). Washing may consist of a single washing step or multiple washing steps.

Washing step d) preferably comprises bringing the BLG crystals into contact with the washing liquid without completely dissolving the BLG crystals and then separating the remaining BLG crystals from the washing liquid.

The washing liquid is preferably chosen to avoid complete dissolution of the BLG crystals and may, for example, contain or even consist essentially of cold demineralized water, cold tap water or cold reverse osmosis filtrate.

The washing liquid may have a pH in the range of 5-6, preferably in the range of 5.0-6.0, and even more preferably in the range of 5.1-6.0, such as in the range of 5.1-5.9.

The washing liquid may have a conductivity of at most 0.1 mS/cm, preferably at most 0.02 mS/cm, and even more preferably at most 0.005 mS/cm.

Flushing fluids with even lower conductivity values can be used. For example, the washing liquid may have a conductivity of not more than 1 mS/cm. Alternatively, the washing liquid may have a conductivity of at most 0.1 mS/cm, such as approx. 0.05 mS/cm.

The washing step is preferably carried out at a low temperature in order to limit the dissolution of crystallized BLG. The temperature of the washing liquid is preferably at most 30°C, more preferably at most 20°C, and even more preferably at most 10°C.

For example, the washing step can be carried out at not more than 5°C, more preferably at not more than 2°C, for example, at approx. 0°C. Temperatures below 0° C. can be used as long as the washing liquid does not freeze at this temperature, for example, due to the presence of one or more substances that lower the freezing point of the freezing point.

In some embodiments of the present invention, the washing liquid contains BLG, for example, in an amount of at least 1% wt./wt., and preferably in an amount of at least 3% wt./wt., such as, for example, in an amount of 4% wt. ./weight.

During the stage of washing d) is usually dissolved no more than 80% wt./weight. from the initial number of crystals BLG, preferably not more than 50% wt./weight. and even more preferably not more than 20%

- 18 040134 w/w from the initial number of BLG crystals. Preferably during the stage of washing d) dissolves no more than 15% wt./weight. from the initial number of BLG crystals, more preferably not more than 10% wt./weight. and even more preferably not more than 5% wt./weight. initial number of crystals

BLG.

The ratio by weight of the total amount of washing liquid and the initial amount of separated BLG crystals is often at least 1, preferably at least 2, and more preferably at least 5. For example, the weight ratio of the amount of washing liquid and the initial amount of separated BLG crystals can be at least 10. Alternatively, the ratio by weight of the total amount of washing liquid and the initial amount of separated BLG crystals can be at least 20, for example, at least 50 or at least 100.

The term total amount of washing liquid refers to the total amount of washing liquid used during the entire process.

In some preferred embodiments of the present invention, one or more washing sequences occur in the same filter device, or in a similar filter device, where the BLG crystals are separated. To the filter cake, mainly containing BLG crystals, the washing liquid is added one or more times in a certain sequence, which is removed through the filter, while the rest of the BLG crystals remain in the filter cake.

In particularly preferred embodiments of the present invention, step c) separation is carried out with a filter that retains BLG crystals. The filter cake is then brought into contact with one or more washing liquids which pass through the filter cake and the filter. It is often preferred that the amount of washing liquid in each case is no more than 10 times the volume of the filter cake, preferably no more than 5 times the volume of the filter cake, more preferably no more than 1 times the volume of the filter cake, still more preferably not more than 0.5 times the volume of the filter cake, such as not more than 0.2 times the volume of the filter cake. The volume of the filter cake contains both solids and liquids (liquids and gases) of the filter cake. The filter cake is preferably washed in this way at least 2 times, preferably at least 4 times and even more preferably at least 6 times.

The washing liquid used in step d) can, for example, be recycled into the milk whey protein raw material or the milk whey protein solution, where the washed BLG can be recovered again.

In addition, the method may include step e), which includes a recrystallization step comprising dissolving the separated BLG crystals in a recrystallization liquid;

adjusting the properties of the liquid for recrystallization to achieve supersaturation by BLG;

crystallization of BLG in a supersaturated recrystallization liquid with corrected properties; and separating the BLG crystals from the remaining corrected recrystallization liquid.

Step e) may involve either a single recrystallization sequence or multiple recrystallization sequences.

In some embodiments of the present invention, the BLG crystals from either step c) or step d) are recrystallized at least 2 times. For example, BLG crystals can be recrystallized at least 3 times, eg at least 4 times.

The washing and recrystallization steps can be combined in any order and can be carried out multiple times as needed.

The BLG crystals separated in step c) can, for example, be subjected to a sequence of the following technological processes:

one or more washing steps (step d) followed by;

one or more recrystallization steps (step e).

Alternatively, the BLG crystals separated in step c) can be processed using the process sequence:

one or more recrystallization steps (step e) followed by;

one or more washing steps (step d).

It is also possible to combine several stages of washing and recrystallization, for example, in the following sequence:

one or more washing steps (step d);

one or more recrystallization steps (step e);

one or more washing steps (step d); And

- 19 040134 one or more stages of recrystallization (stage e).

Or, for example, in this order:

one or more recrystallization steps (step e);

one or more washing steps (step d);

one or more recrystallization steps (step e);

one or more washing steps (step d).

In some embodiments of the present invention, the method further comprises subjecting the separated BLG to additional BLG enrichment steps, such as based on chromatography or selective filtration. However, in other preferred embodiments of the present invention, the process does not include additional BLG enrichment steps after step b). By the term BLG further enrichment step is meant the method step by which BLG is enriched relative to the total amount of protein, and the step does not refer to crystallization of BLG or processing of BLG crystals. An example of such a BLG further enrichment step is ion exchange chromatography. Washing the BLG crystals and/or recrystallizing the BLG is not considered an additional BLG enrichment step.

In some particularly preferred embodiments of the present invention, the method comprises a drying step f) wherein the BLG-containing composition obtained in steps b), c), d) or e) is converted into a dry composition.

In the context of the present invention, the term dry means that the composition or product of interest contains no more than 6% w/w. water and preferably even less.

In the context of the present invention, the term BLG-containing composition is used to describe a composition that is subjected to drying step f).

In the context of the present invention, a BLG-containing composition obtained in steps b), c), d) or e) means a composition that contains at least some of the BLG from step b), c), d) or e). In some preferred embodiments of the present invention, the BLG-containing composition obtained in steps b), c), d) or e) is directly obtained in steps b), c), d) or e). However, in other preferred embodiments of the present invention, the BLG-containing composition obtained in steps b), c), d) or e) is the result of additional processing of the composition obtained directly in steps b), c), d) or e).

It is often preferred that the BLG-containing composition contains a significant amount of the BLG present in the composition obtained directly from steps b), c), d) or e). In some preferred embodiments, the implementation of the present invention containing BLG composition obtained in stages b), c), d) or e), contains at least 50% wt./weight. BLG obtained in steps b), c), d) or e), preferably at least 70% and even more preferably at least 80%.

Preferably containing BLG composition obtained in stages b), c), d) or e), contains at least 85% wt./weight. BLG obtained in steps b), c), d) or e). More preferably containing BLG composition obtained in stages b), c), d) or e), contains at least 90% wt./weight. BLG obtained in steps b), c), d) or e). Even more preferably containing BLG composition obtained in stages b), c), d) or e), contains at least 95% wt./weight. BLG obtained in steps b), c), d) or e). Most preferably, the BLG-containing composition obtained in steps b), c), d) or e) contains 100% w/w. BLG obtained in steps b), c), d) or e).

In some preferred embodiments of the present invention, the drying step includes one or more of spray drying, lyophilization drying, spin drying, rotary drum drying, and/or fluid bed drying.

In some particularly preferred embodiments of the present invention, the drying step comprises a BLG-containing composition in which the BLG crystal has been dissolved and the resulting powder does not contain the BLG crystals formed in step b) or by recrystallization prior to the drying step. These embodiments are preferred if the BLG food composition is to resemble, for example, conventional dried whey protein powder.

For example, BLG crystals can be dissolved by raising the temperature;

increasing conductivity, for example by adding one or more salts;

changes in pH, for example, not within the range of 5-6;

reducing the concentration of BLG, for example, by dilution;

or combinations of the above.

Spray drying is in this case the preferred method of drying a BLG-containing composition that does not contain BLG crystals.

In other particularly preferred embodiments of the present invention, the drying step comprises a BLG-containing composition that still contains BLG crystals,

- 20 040134 and where the resulting powder contains BLG crystals. These embodiments are preferred if the BLG food composition is to have a higher density than conventional dried whey protein powder.

In some particularly preferred embodiments of the present invention, the drying step comprises a BLG-containing composition that still contains BLG crystals, and wherein the resulting powder contains BLG crystals. These embodiments are preferred if the BLG food composition is to have a higher density than conventional dried whey protein powder.

Example 7 states that the present inventors have found that spray drying a suspension of BLG crystals while retaining at least some of the crystal structure is possible if the dried BLG crystals are resuspended in cold demineralized water. It is particularly preferred to avoid exposing the BLG-containing composition containing BLG crystals to a heat treatment regimen that dissolves a significant amount of the BLG crystals prior to spraying. Thus, if preheating a BLG-containing composition containing BLG crystals is used prior to spraying, it is preferable to carefully control the heat load.

In some embodiments of the present invention, the BLG-containing composition containing BLG crystals has a temperature of no more than 70°C at the outlet of the spray device (eg, nozzle or atomizer), preferably no more than 60°C, more preferably no more than 50°C. In some preferred embodiments of the present invention, the BLG-containing composition containing BLG crystals has a temperature of no more than 40° C. at the outlet of the spray device, preferably no more than 30° C., more preferably no more than 20° C., even more preferably no more than 10° C. C and most preferably not more than 5°C.

The spray device of a spray dryer is a device, such as a nozzle or an atomizer, which converts the solution or suspension to be dried into droplets that enter the drying chamber of the spray dryer.

It is particularly preferred that the BLG-containing composition containing BLG crystals has a temperature in the range of 0-50°C at the outlet of the spray device, preferably in the range of 2-40°C, more preferably in the range of 4-35°C and most preferably in the range 5-10°C at the outlet of the spray device.

In some preferred embodiments of the present invention, the BLG-containing composition has a BLG crystallinity of at least 20% at the outlet of the spray device, preferably at least 40%, more preferably at least 60%, even more preferably at least 80 % and most preferably at least 90%, such as preferably 97-100%. The BLG-containing composition may either be a BLG isolate, eg, contain BLG in an amount greater than 90% w/w. relative to the total protein, or it may contain significant amounts of other proteins and therefore contain BLG in an amount of not more than 90% wt./weight. relative to total protein.

In some preferred embodiments of the present invention, the BLG-containing composition may have a protein composition in the form of conventional liquid WPC or WPI, or conventional liquid SPC or SPI, as described herein, but have at least 20% BLG crystallinity at the outlet of the spray gun. device, preferably at least 40%, more preferably at least 60%, even more preferably at least 80% and most preferably at least 90%, such as preferably 97-100%.

The inlet gas temperature of the spray dryer is preferably in the range of 140-220°C, more preferably in the range of 160-200°C, and even more preferably in the range of 170-190°C, such as preferably about 180°C. The gas temperature at the exit of the spray dryer is preferably in the range of 50-95°C, more preferably in the range of 70-90°C and even more preferably in the range of 80-88°C, such as preferably about 85°C. In general, solids that are subjected to spray drying are considered to be heated to a temperature that is 10-15° C. less than the outlet gas temperature.

In some preferred embodiments of the present invention, the temperature in the spray dryer is preferably in the range of 50-85°C, more preferably in the range of 60-80°C, and even more preferably in the range of 65-75°C, such as preferably approximately 70° C.

The idea of spray drying a suspension of BLG crystals has not been disclosed in the prior art and is in itself a separate aspect of the present invention.

Thus, an aspect of the present invention relates to a process for preparing a spray-dried food powder composition containing BLG, said composition comprising dried BLG crystals, the method comprising the steps of providing a liquid BLG-containing composition containing BLG crystals, and preferably having the degree of crystallinity BLG constituting at least 20%, while the specified liquid containing BLG composition preferably contains at least 10% wt./weight. of the total solids and preferably contains at least 5% wt./weight. BLG; and atomizing the liquid BLG-containing composition in a drying chamber of an operating spray dryer to convert the liquid BLG-containing composition containing the BLG crystals into a powder.

In some preferred embodiments of the present invention, the BLG-containing composition to be dried is mixed with the dry BLG isolate to raise the solids content to a level where the mixture can be dried by fluid bed drying. This is also referred to as back blending and allows highly cost-effective drying of the BLG based product. These embodiments are particularly preferred for BLG-containing compositions that contain BLG crystals.

An advantage of the process of the present invention is that the BLG-containing composition to be dried can have a very high solids content prior to the drying step, and thus less water needs to be removed and less energy is consumed during the drying process step.

In some preferred embodiments of the present invention, the BLG-containing composition obtained in steps b), c), d) or e) is characterized by a solids content of at least 20% w/w. Preferably containing BLG composition obtained in stages b), c), d) or e), is characterized by a solids content of at least 30% wt./weight. More preferably, the BLG-containing composition obtained in steps b), c), d) or e) has a solids content of at least 40% w/w. Even more preferably, the BLG-containing composition obtained in steps b), c), d) or e) has a solids content of at least 50% w/w, such as at least 60% wt. /weight.

In other preferred embodiments, the implementation of the present invention containing BLG composition obtained in stages b), c), d) or e), is characterized by a solids content in the range of 20-80% wt./weight. Preferably containing BLG composition obtained in stages b), c), d) or e), is characterized by a solids content in the range of 30-70% wt./weight. More preferably containing BLG composition obtained in stages b), c), d) or e), is characterized by a solids content in the range of 40-65% wt./weight. Even more preferably, the BLG-containing composition obtained in steps b), c), d) or e) has a solids content in the range of 50-65% w/w, such as eg approx. 60% w/w

The present inventors have found that the higher the degree of crystallinity of the BLG-containing composition, the less water is bound to the BLG-containing composition and the higher the total solids content of the BLG-containing composition can be achieved prior to the drying step.

Thus, in some preferred embodiments of the present invention, the BLG-containing composition is characterized by a degree of BLG crystallinity of at least 10% w/w. Preferably, the BLG in the BLG-containing composition has a degree of crystallinity of at least 20% w/w. More preferably, the BLG in the BLG-containing composition has a degree of crystallinity of at least 30% w/w. Even more preferably, the BLG in the BLG-containing composition has a degree of crystallinity of at least 40% w/w.

Even higher degrees of crystallinity are often preferred. Thus, in some preferred embodiments of the present invention, the BLG in the BLG-containing composition has a degree of crystallinity of at least 50% w/w. Preferably, the BLG in the BLG-containing composition has a degree of crystallinity of at least 60% w/w. More preferably, the BLG from the BLG-based food composition has a degree of crystallinity of at least 70% w/w. Even more preferably, the BLG in the BLG-containing composition has a degree of crystallinity of at least 80% w/w. Most preferably, the BLG in the BLG-containing composition has a degree of crystallinity of at least 90% w/w, preferably at least 95% w/w, more preferably at least 97% w/w. and even more preferably at least 99% wt./weight.

The authors found that a reduced water content, as a rule, increases the degree of crystallinity of BLG in the composition. Thus, compositions with a high water:BLG ratio (e.g., a 4% suspension of BLG crystals in water) tend to have lower BLG crystallinity than compositions that have a lower water:BLG ratio (e.g., filter cake or wet isolated crystals) under the same conditions.

- 22 040134

The method of the present invention can be carried out using moderate temperatures that do not compromise the nutritional value of either BLG or other milk whey proteins of the milk whey protein solution.

In some preferred embodiments of the present invention, the BLG is not exposed to temperatures above 90°C during this method. Preferably, the BLG is not exposed to temperatures above 80° C. during this process. Even more preferably, the BLG is not exposed to temperatures above 75° C. during this process. It should be noted that while spray drying often uses temperatures in excess of 150°C, the short exposure time and simultaneous evaporation of water means that spray-dried proteins are not subjected to temperatures above 50-70°C.

The authors found evidence that prolonged heating during the drying step reduces the amount of BLG that is present in crystalline form. In some preferred embodiments of the present invention, the exposure to heat during the drying step is kept low enough to ensure that the degree of BLG denaturation is no more than 10%, preferably no more than 4%, more preferably no more than 1%, even more preferably no more than 0.4%, and even more preferably not more than 0.1%. Most preferably, the drying step does not result in detectable denaturation of the BLG at all.

The degree of denaturation due to the drying step is calculated by determining the BLG content (relative to total solids) of the BLG-based composition to be dried (from _{to step} f) in step f) and the BLG content (relative to total solids) of re-dissolved dried composition using the formula:

^{Degree of denaturation ((s} to stage f ^- s after stage 1 ^)/s to stage f ^{) x 100} %

Some preferred embodiments of the present invention relate to a method for preparing a food composition containing beta-lactoglobulin (BLG) in crystalline form, the method comprising the steps of:

a) providing a milk whey protein solution comprising BLG and at least one additional milk whey protein, wherein said milk whey protein solution is supersaturated with BLG and has a pH in the range of 5-6, wherein said milk whey protein solution contains

70-100% w/w protein relative to total solids;

30-90% w/w BLG relative to the total amount of protein and preferably 30-70% wt./weight. BLG;

4-50% w/w ALA relative to the total amount of protein and preferably 8-35% wt./weight. ALA;

0-25% w/w CMP for protein;

at least 10% wt./weight. protein relative to the total weight of the milk whey protein solution,

b) crystallizing the BLG in a supersaturated whey protein solution, preferably by adding crystallization seeds, and

f) drying the BLG-containing composition which is obtained directly from step b), said BLG-containing composition preferably having a BLG crystallinity of at least 30%, the process omitting steps c), d) or e).

The milk whey protein solution is preferably a demineralized milk whey protein solution and is characterized by a preferred conductivity to total protein ratio of not more than 0.3 and/or an ultrafiltrate conductivity of not more than 7 mS/cm.

In these embodiments, the BLG crystals are not separated from the whey protein solution, but dried to produce a high density BLG food composition in powder form.

In addition, the present invention relates to food compositions obtained using these embodiments.

Other preferred embodiments of the present invention relate to a method for preparing a food composition containing beta-lactoglobulin (BLG) in crystallized form, the method comprising the steps of:

a) providing a milk whey protein solution comprising BLG and at least one additional milk whey protein, wherein said milk whey protein solution is supersaturated with BLG and has a pH in the range of 5-6, wherein said milk whey protein solution contains:

70-100% w/w protein relative to total solids;

30-90% w/w BLG relative to total protein and preferably 30-70%;

4-50% w/w ALA relative to total protein and preferably 8-35%;

- 23 040134

0-25% w/w CMP relative to total protein;

at least 10% wt./weight. protein relative to the total weight of the milk whey protein solution,

b) crystallizing the BLG in a supersaturated whey protein solution, preferably by adding crystallization seeds,

c) separating the BLG crystals from the rest of the milk whey protein solution,

d) optionally washing the separated BLG crystals obtained in step c),

e) optionally recrystallizing the BLG crystals obtained in step c) or d), and

f) drying the BLG-containing composition obtained in and preferably directly obtained in steps c), d) or e), wherein the BLG-containing composition contains BLG crystals and preferably has a BLG crystallinity of at least 30%.

The milk whey protein solution is preferably a demineralized milk whey protein solution and is characterized by a preferred conductivity to total protein ratio of not more than 0.3 and/or an ultrafiltrate conductivity of not more than 7 mS/cm.

These embodiments are particularly useful for producing low mineral and low phosphorus BLG food compositions as high density powders.

In addition, the present invention relates to food compositions obtained using these embodiments.

Another preferred embodiment of the present invention relates to a method for preparing a food composition containing beta-lactoglobulin in isolated form, the method comprising the steps of:

a) providing a milk whey protein solution comprising BLG and at least one additional milk whey protein, wherein said milk whey protein solution is supersaturated with BLG and has a pH in the range of 5-6, wherein said milk whey protein solution contains:

70-100% w/w protein relative to total solids;

30-90% w/w BLG relative to the total amount of protein and preferably 30-70% wt./weight. BLG;

5-50% w/w ALA relative to the total amount of protein and preferably 8-35% wt./weight. ALA;

0-25% w/w CMP relative to total protein;

at least 10% wt./weight. protein relative to the total weight of the milk whey protein solution,

b) crystallizing the BLG in a supersaturated whey protein solution, preferably by adding crystallization seeds,

c) separating the BLG crystals from the rest of the milk whey protein solution,

d) optionally washing the separated BLG crystals obtained in step c),

e) optionally recrystallizing the BLG crystals obtained in step c) or d), and

f) drying the BLG-containing composition obtained in steps c), d) or e), wherein the BLG-containing composition does not contain BLG crystals.

The milk whey protein solution is preferably a demineralized milk whey protein solution and is characterized by a preferred conductivity to total protein ratio of not more than 0.3 and/or an ultrafiltrate conductivity of not more than 7 mS/cm.

In these embodiments, the BLG crystals are dissolved before drying.

In addition, the present invention relates to food compositions obtained using these embodiments.

In some preferred embodiments, the process of the present invention is carried out as a batch process. Alternatively, and sometimes preferably, the process can be carried out as a semi-continuous process. In other preferred embodiments, the process is carried out as a continuous process.

The advantage of the present method is that it is much faster than comparative prior art BLG crystallization methods. The duration from the initial adjustment of the milk whey protein raw material to the completion of the separation in step c may be no more than 10 hours, preferably no more than 4 hours, more preferably no more than 2 hours, and even more preferably no more than 1 hour.

An additional aspect of the present invention relates to an isolated BLG crystal obtained using the method described in this document.

In the context of the present invention, the term isolated BLG crystal refers to a BLG crystal that has been separated from the solution in which it was formed, but which may still contain internal water, i. water hydrating the BLG molecules of the crystal.

The isolated BLG crystal preferably has an orthorhombic space group

P 21 21 21.

Preferably, the isolated BLG crystal has a rhombic space group P 21 21 21 and unit cell parameters a=68.68 (±5%) A, b=68.68 (±5%) A and c=156.65 (±5%) A; and has integral unit cell angles α=90° (±2%), β=90° (±2%) and γ=90° (±2%).

In some preferred embodiments of the present invention, the isolated BLG crystal has a rhombic space group P 2 ₁ 2 ₁ 2 ₁ and unit cell parameters a=68.68 (±2%) A, b=68.68 (±2%) A and c =156.65 (±2%) A; and has integral unit cell angles α=90° (±1%), β=90° (±1%) and γ=90° (±1%).

Even more preferably, the isolated BLG crystal may have a rhombic space group P 21 21 21 and unit cell parameters a=68.68 (±1%) A, b=68.68 (±1%) A and c=156.65 (± 1%) A; and have integral unit cell angles α=90° (±0.5%), β=90° (±0.5%) and γ=90° (±0.5%).

Most preferably, the isolated BLG crystal has a rhombic space group P 2 ₁ 2 ₁ 2 ₁ and unit cell parameters a=68.68 A, b=68.68 A and c=156.65 A; and has integral unit cell angles α=90°, β=90° and γ=90°.

Selected crystal BLG may, for example, contain at least 20% wt./weight. BLG and not more than 80% wt./wt. water. Preferably, the isolated BLG crystal may contain at least 40% w/w. BLG and water in the range of 0-60% wt./wt. Even more preferably, the isolated BLG crystal contains BLG in the range of 40-60% w/w. and water in the range from about 40 to about 60% wt./weight.

The present inventors have found that the BLG crystals of the present invention unexpectedly have the ability to restore their original crystal structure after drying and rehydration. This is particularly advantageous in applications where the crystal structure provides certain advantages of BLG.

Another aspect of the present invention relates to a food composition containing beta-lactoglobulin, for example, to a food composition obtained using the method defined in this document.

Another aspect of the present invention relates to a food composition based on BLG containing at least 90% wt./weight. BLG relative to total solids. Such a BLG-based food composition can be obtained using the method defined herein.

An additional aspect of the present invention relates to a food composition based on BLG containing dried crystals of BLG, at least 20% wt./weight. BLG relative to total solids, and preferably having a BLG crystallinity of at least 20%. Such a BLG-based food composition containing dried BLG crystals can be prepared using the method defined herein.

In some preferred embodiments of the present invention, the BLG in the BLG food composition has a lactosylation degree of at most 1. Preferably, the BLG in the BLG food composition has a lactosylation degree of at most 0.6. More preferably, the BLG in the BLG food composition has a lactosylation degree of at most 0.4. Even more preferably, the BLG in the BLG food composition has a lactosylation degree of at most 0.2. Most preferably, the BLG in the BLG food composition has a degree of lactosylation of at most 0.1, such as preferably at most 0.01.

In some preferred embodiments of the present invention, the BLG in the BLG-based food composition contains at least 90% w/w. non-lactosylated BLG, preferably at least 95% w/w. non-lactosylated BLG and even more preferably at least 98% wt./weight. non-lactosylated BLG.

The percentage of non-lactosylated BLG is determined according to Example 9.1.

In some preferred embodiments of the present invention, the BLG in the BLG-based food composition has a degree of crystallinity of at least 10% w/w. Preferably, the BLG in the BLG-based food composition has a degree of crystallinity of at least 20% w/w. More preferably, the BLG in the BLG-based food composition has a degree of crystallinity of at least 30% w/w. Even more preferably, the BLG in the BLG-based food composition has a degree of crystallinity of at least 40% w/w.

Even higher degrees of crystallinity are often preferred. Thus, in some preferred embodiments of the present invention, the BLG in the BLG food composition has a degree of crystallinity of at least 50%.

- 25 040134 w/w Preferably, the BLG in the BLG-based food composition has a degree of crystallinity of at least 60% w/w. More preferably, the BLG in the BLG-based food composition has a degree of crystallinity of at least 70% w/w. Even more preferably, the BLG in the BLG-based food composition has a degree of crystallinity of at least 80% w/w. Most preferably, the BLG in the BLG-based food composition has a degree of crystallinity of at least 90% w/w. and preferably at least 95% wt./weight.

The degree of crystallinity of BLG in a liquid with a pH in the range of 5-6 is measured according to example 9.7. The degree of crystallinity of BLG in powdered material is measured according to example 9.8. If the food composition is a dry product, but not in the form of a powder, it should be reduced to powder, for example, by grinding or milling, before exposure using the method of example 9.8.

In some preferred embodiments of the present invention, the BLG food composition is WPC, WPI, SPC, or SPI in which at least a portion of the BLG is present in crystalline form. Food composition based on BLG may, for example, contain no more than 90% wt./weight. BLG relative to total protein and is characterized by a BLG crystallinity of at least 10%. For example, a food composition based on BLG may contain no more than 80% wt./weight. BLG relative to total protein and have a BLG crystallinity of at least 10%. For example, a food composition based on BLG may contain 30-70% wt./weight. BLG relative to total protein and have a BLG crystallinity of at least 10%.

In other preferred embodiments of the present invention, the BLG-based food composition contains no more than 90% w/w. BLG relative to the total amount of protein and is characterized by a degree of BLG crystallinity of at least 30%. Preferably, the BLG-based food composition may contain no more than 80% w/w. BLG relative to total protein and have a BLG crystallinity of at least 30%. Even more preferably, the BLG-based food composition may contain 30-70% w/w. BLG relative to total protein and have a BLG crystallinity of at least 30%.

The present inventors have found that the present invention makes it possible to obtain a whey protein food product with a very low content of phosphorus and other minerals, which is preferable for patients suffering from kidney disease or who have reduced kidney function to some extent.

The BLG food composition is preferably a low phosphorus composition.

In the context of the present invention, the term "low phosphorus" refers to a composition, such as a liquid, powder or other food product, which has a total phosphorus content of not more than 100 mg of phosphorus per 100 g of protein. Preferably, the low phosphorus composition has a total content of no more than 80 mg of phosphorus per 100 g of protein. More preferably, the low phosphorus composition may have a total content of no more than 50 mg of phosphorus per 100 g of protein. Even more preferably, the low phosphorus composition may have a total phosphorus content of not more than 20 mg of phosphorus per 100 g of protein. Even more preferably, the low phosphorus composition may have a total phosphorus content of not more than 5 mg of phosphorus per 100 g of protein. The low phosphorus compositions of the present invention can be used as a food ingredient in the preparation of a food product intended for patient groups with reduced renal function.

Thus, in some particularly preferred embodiments of the present invention, the BLG food composition contains no more than 80 mg of phosphorus per 100 g of protein. Preferably, the BLG food composition contains no more than 30 mg of phosphorus per 100 g of protein. More preferably, the BLG food composition contains no more than 20 mg of phosphorus per 100 g of protein. Even more preferably, the BLG food composition contains no more than 10 mg of phosphorus per 100 g of protein. Most preferably, the BLG food composition contains no more than 5 mg of phosphorus per 100 g of protein.

The phosphorus content refers to the total amount of elemental phosphorus in the composition of interest and is determined according to example 9.5.

In other preferred embodiments of the present invention, the BLG food composition is a low mineral composition.

In the context of the invention, the term low mineral content refers to a composition, such as a liquid, powder or other food product, which is characterized by at least one, preferably two, and even more preferably all of the following:

the content of ash substances is not more than 1.2% wt./weight. relative to the total amount of solids

- 26 040134 companies;

the total content of calcium and magnesium is not more than 0.3% wt./weight. relative to total solids;

the total content of sodium and potassium is not more than 0.10% wt./weight. relative to total solids;

the total phosphorus content is not more than 100 mg of phosphorus per 100 g of protein.

Preferably the low mineral composition is characterized by at least one, preferably two or more, and even more preferably all of the following:

the content of ash substances is not more than 0.7% wt./wt. relative to total solids;

the total content of calcium and magnesium is not more than 0.2% wt./weight. relative to total solids;

the total content of sodium and potassium is not more than 0.08% wt./weight. relative to total solids;

the total phosphorus content is not more than 80 mg of phosphorus per 100 g of protein.

Even more preferably, the low mineral composition is characterized by at least one, preferably two or more, and even more preferably all of the following:

the content of ash substances is not more than 0.5% wt./wt. relative to total solids;

the total content of calcium and magnesium is not more than 0.15% wt./weight. relative to total solids;

the total content of sodium and potassium is not more than 0.06% wt./weight. relative to total solids;

the total phosphorus content is not more than 50 mg of phosphorus per 100 g of protein.

It is particularly preferred that the low mineral composition has the following characteristics:

the ash content is not more than 0.5% wt./weight. relative to total solids;

the total content of calcium and magnesium is not more than 0.15% wt./weight. relative to total solids;

the total content of sodium and potassium is not more than 0.06% wt./weight. relative to total solids;

the total phosphorus content is not more than 50 mg of phosphorus per 100 g of protein.

In some preferred embodiments, the implementation of the present invention in the food composition based on BLG, the total amount of proteins is at least 25% wt./weight. relative to the total solids of the food composition based on BLG. Preferably, in the BLG-based food composition, the total amount of proteins is at least 50% w/w. relative to the total solids of the food composition based on BLG. More preferably, in the BLG-based food composition, the total amount of proteins is at least 75% w/w. relative to the total solids of the food composition based on BLG. Even more preferably, in the food composition based on BLG, the total amount of proteins is at least 90% wt./weight. relative to the total solids of the food composition based on BLG.

In some preferred embodiments of the present invention, the total amount of protein in the BLG food composition is in the range of 25-100% w/w. relative to total solids. Preferably, the total amount of protein in the BLG food composition is in the range of 50-100% w/w. More preferably, the total amount of protein in the BLG food composition is in the range of 75-100% w/w. relative to total solids. Even more preferably, the total amount of protein in the BLG food composition is in the range of 90-100% w/w. relative to total solids.

In some preferred embodiments of the present invention, the BLG-based food composition contains at least 75% w/w. BLG relative to total protein. Preferably, the BLG-based food composition may contain at least 90% w/w. BLG relative to total protein. More preferably, the BLG-based food composition may contain at least 95% w/w. BLG relative to total protein. Even more preferably, the BLG-based food composition may contain at least 97% w/w. BLG relative to total protein. Most preferably, the BLG-based food composition may contain approx. 100% w/w BLG relative to total protein.

In some preferred embodiments of the present invention, the BLG-based food composition contains no more than 10% w/w. carbohydrates, preferably not more than 5% wt./weight.

- 27 040134 carbohydrates, more preferably not more than 1% wt./weight. carbohydrates and even more preferably not more than 0.1% wt./weight. carbohydrates.

The BLG food composition may also contain lipids, for example in the form of triglycerides and/or other types of lipids such as phospholipids.

In some embodiments, implementation of the present invention in the food composition based on BLG, the total amount of lipids is not more than 1% wt./weight. relative to total solids. Preferably, in the BLG-based food composition, the total amount of lipids is not more than 0.5% w/w. relative to total solids. More preferably, in the BLG-based food composition, the total amount of lipids is not more than 0.1% w/w. relative to total solids. Even more preferably, in the BLG-based food composition, the total amount of lipids is not more than 0.05% w/w. relative to total solids. Most preferably, in the BLG-based food composition, the total amount of lipids is not more than 0.01% w/w. relative to total solids.

In some preferred embodiments of the present invention, the BLG food composition is a dry composition and, for example, a powder. It is particularly preferred that the BLG food composition is a spray dried powder.

The present inventors observed that powdered BLG food compositions in which at least some of the BLG was in crystalline form after drying had a higher density than a comparative BLG composition without BLG crystals (see Example 7). This high density effect, surprisingly, has also been noted in powdered BLG food compositions, which are prepared from suspensions of spray-dried BLG crystals.

Thus, in some preferred embodiments of the present invention, the powdered BLG food composition has a bulk density of at least 0.40 g/mL. Preferably, the powdered BLG food composition has a bulk density of at least 0.45 g/ml. More preferably, the powdered BLG food composition has a bulk density of at least 0.50 g/ml. Even more preferably, the powdered BLG food composition has a bulk density of at least 0.6 g/ml. For example, a powdered BLG food composition may have a bulk density of at least 0.7 g/mL.

The advantage in bulk density extends to both BLG food composition powders in which BLG is essentially the only protein present, and BLG food composition powders in which the concentration of BLG has not been increased relative to other proteins that were present in the milk whey protein solution. . Thus, the present invention provides high density powders of both isolated BLG and crude milk whey protein that contain significant amounts of ALA and other milk whey proteins in addition to BLG.

In some preferred embodiments of the present invention, the BLG-based food composition in powder form has a bulk density of at least 0.45 g/ml and contains at least 70% w/w. protein relative to the total weight of the composition. More preferably, the BLG-based food composition in powder form has a bulk density of at least 0.50 g/ml and contains at least 70% w/w. protein relative to the total weight of the composition. Even more preferably, the powdered BLG-based food composition has a bulk density of at least 0.6 g/ml and contains at least 70% w/w. protein relative to the total weight of the composition. Food composition based on BLG in the form of a powder, characterized by a bulk density of at least 0.7 g/ml and contains at least 70% wt./weight. protein relative to the total weight of the composition.

In other preferred embodiments of the present invention, the BLG-based food composition in powder form is characterized by a bulk density of at least 0.45 g/ml and contains at least 80% w/w. protein relative to the total weight of the composition. More preferably, the BLG-based food composition in powder form has a bulk density of at least 0.50 g/ml and contains at least 80% w/w. protein relative to the total weight of the composition. Even more preferably, the powdered BLG-based food composition has a bulk density of at least 0.6 g/ml and contains at least 80% w/w. protein relative to the total weight of the composition. The BLG-based food composition in powder form has a bulk density of at least 0.7 g/ml and contains at least 80% w/w. protein relative to the total weight of the composition.

For example, a powdered BLG-based food composition may have a bulk density in the range of 0.40-1.5 g/ml and contain at least 80% w/w. protein relative to the total weight of the composition. Preferably, the powdered BLG food composition has a bulk density in the range of 0.45-1.0 g/ml and contains at least 80% w/w. protein relative to the total weight of the composition. More preferably, the BLG-based powdered food composition may have a bulk density in the range of 0.50-0.9 g/ml and contain at least 80% w/w. protein relative to the total weight of the composition. Even more preferably, the BLG powdered food composition has a bulk density in the range of 0.6-0.9 g/ml and contains at least 80% w/w. protein relative to the total weight of the composition. For example, powdered food composition based on BLG may have a bulk density in the range of 0.6-0.8 g/ml and contains at least 80% wt./weight. protein relative to the total weight of the composition.

The authors have found that the high density powders of the present invention preferably allow for more cost effective powder packaging and logistics since less packaging material is needed per kilogram of powder and more powder (by weight) can be transported by a specific container or vehicle.

For example, a powdered BLG food composition may have a bulk density in the range of 0.40-1.5 g/mL. Preferably, the powdered BLG food composition has a bulk density in the range of 0.45-1.0 g/ml. More preferably, the powdered BLG food composition may have a bulk density in the range of 0.50-0.9 g/mL. Even more preferably, the BLG powdered nutritional composition has a bulk density in the range of 0.6-0.9 g/mL. For example, a powdered BLG food composition may have a bulk density in the range of 0.60.8 g/mL.

In other preferred embodiments of the present invention, the powdered BLG food composition has a bulk density in the range of 0.50-1.5 g/ml. Preferably, the powdered BLG food composition has a bulk density in the range of 0.55-1.0 g/ml. More preferably, the powdered BLG food composition may have a bulk density in the range of 0.60-1.0 g/mL. Even more preferably, the BLG powdered nutritional composition has a bulk density in the range of 0.65-1.0 g/mL. The powdered BLG food composition may preferably have a bulk density in the range of 0.70-1.0 g/ml.

For example, a powdered BLG-based food composition may have a bulk density in the range of 0.40-1.5 g/ml and contain at least 70% w/w. protein relative to the total weight of the composition. Preferably, the BLG-based powdered food composition has a bulk density in the range of 0.45-1.0 g/ml and contains at least 70% w/w. protein relative to the total weight of the composition. More preferably, the BLG-based powdered food composition may have a bulk density in the range of 0.50-0.9 g/ml and contain at least 70% w/w. protein relative to the total weight of the composition. Even more preferably, the BLG powdered food composition has a bulk density in the range of 0.6-0.9 g/ml and contains at least 70% w/w. protein relative to the total weight of the composition. For example, powdered food composition based on BLG may have a bulk density in the range of 0.6-0.8 g/ml and contains at least 70% wt./weight. protein relative to the total weight of the composition.

For example, a powdered BLG-based food composition may have a bulk density in the range of 0.40-1.5 g/ml and contain at least 80% w/w. protein relative to the total weight of the composition. Preferably, the BLG-based powdered food composition has a bulk density in the range of 0.45-1.0 g/ml and contains at least 80% w/w. protein relative to the total weight of the composition. More preferably, the BLG-based powdered food composition may have a bulk density in the range of 0.50-0.9 g/ml and contain at least 80% w/w. protein relative to the total weight of the composition. Even more preferably, the BLG powdered food composition has a bulk density in the range of 0.6-0.9 g/ml and contains at least 80% w/w. protein relative to the total weight of the composition. For example, powdered food composition based on BLG may have a bulk density in the range of 0.6-0.8 g/ml and contains at least 80% wt./weight. protein relative to the total weight of the composition.

In other preferred embodiments of the present invention, the food composition based on BLG in the form of a powder, characterized by a bulk density in the range of 0.50-1.5 g/ml and contains at least 70% wt./weight. protein relative to the total weight of the composition. Preferably, the BLG-based powdered food composition has a bulk density in the range of 0.55-1.0 g/ml and contains at least 70% w/w. protein relative to the total weight of the composition. More preferably, the BLG-based powdered food composition may have a bulk density in the range of 0.60-1.0 g/ml and contain at least 70% w/w. protein relative to the total weight of the composition. Even more preferably, the powdered BLG food composition has a bulk density in the range of 0.65-1.0 g/ml and contains at least 70% w/w. protein relative to the total weight of the composition. Powdered food composition based on BLG may preferably be characterized by a bulk density in the range of 0.70-1.0 g/ml and contains at least 70% wt./weight. protein relative to the total weight of the composition.

In other preferred embodiments of the present invention, the BLG-based food composition in powder form has a bulk density in the range of 0.50-1.5 g/ml and contains at least 80% w/w. protein relative to the total weight of the composition. Preferably, the BLG-based powdered food composition has a bulk density in the range of 0.55-1.0 g/ml and contains at least 80% w/w. protein relative to the total weight of the composition. More preferably, the BLG-based powdered food composition may have a bulk density in the range of 0.60-1.0 g/ml and contain at least 80% w/w. protein relative to the total weight of the composition. Even more preferably, the powdered food composition based on BLG was characterized by a bulk density in the range of 0.65-1.0 g/ml and contained at least 80% wt./weight. protein relative to the total weight of the composition. Powdered food composition based on BLG may preferably be characterized by a bulk density in the range of 0.70-1.0 g/ml and contains at least 80% wt./weight. protein relative to the total weight of the composition.

The bulk density of the powder is measured according to example 9.3.

The inventors have found indications that the BLG compositions of the present invention have better long-term stability than similar BLG compositions. This is particularly the case when at least some of the BLG is present as BLG crystals, which appear to provide better storage stability of the BLG molecules.

In some preferred embodiments of the present invention, the BLG dry composition has a furosin content of no more than 80 mg/100 g of protein after 60 days at 30°C, preferably no more than 60 mg/100 g of protein, more preferably no more than 40 mg /100 g of protein and even more preferably not more than 20 mg/100 g of protein. Most preferably, the BLG dry composition has a furosin content of at most 10 mg/100 g of protein after 60 days at 30°C.

In some preferred embodiments of the present invention, the BLG dry composition has a furosin content of at most 80 mg/100 g of protein, preferably at most 60 mg/100 g of protein, more preferably at most 40 mg/100 g of protein, and even more preferably not more than 20 mg/100 g of protein. Most preferably, the dry BLG composition has a furosin content of at most 10 mg/100 g of protein. Preferably the dry BLG composition has a furosin content of 0 mg/100 g protein.

In some preferred embodiments of the present invention, the BLG in the BLG dry composition has a degree of lactosylation of at most 1 after 60 days at 30° C., preferably at most 0.6, more preferably 0.2, even more preferably at most 0, 1 and most preferably not more than 0.01.

In some preferred embodiments of the present invention, the BLG food composition is a liquid composition. Liquid food composition based on BLG preferably contains at least 20% wt./weight. water, more preferably at least 30% wt./weight. water, even more preferably at least 40% wt./weight.

For example, a BLG liquid food composition may contain water in the range of 20-90% w/w, more preferably water in the range of 30-80% w/w, even more preferably at least 40% w/w. .

The present inventors have found that the BLG food compositions of the present invention have an unexpectedly low degree of protein denaturation, although spray drying was used to produce a BLG food powder composition (see Example 11).

Thus, in some preferred embodiments of the present invention, the BLG food composition is characterized by a degree of protein denaturation of no more than 2%. Preferably, the BLG food composition has a protein denaturation of at most 1.5%. More preferably, the BLG food composition has a protein denaturation of at most 1.0%. Even more preferably, the BLG food composition has a protein denaturation degree of at most 0.8%. Even more preferably, the BLG food composition has a protein denaturation of at most 0.5%.

In some preferred embodiments of the present invention, the BLG food composition is a dry powder, and preferably a spray-dried powder, and has a protein denaturation of not more than 2%, and preferably not more than 1.5%. More preferably, the BLG dry food composition, eg in the form of a spray-dried powder, has a protein denaturation of at most 1.0%. Even more preferably, the BLG dry food composition, for example in the form of a spray-dried powder, has a protein denaturation of at most 0.8%. Even more preferably, the BLG dry food composition, for example in the form of a spray-dried powder, has a protein denaturation of at most 0.5%.

In some preferred embodiments of the present invention, the BLG-based food composition contains no more than 6% w/w. water;

at least 80% total protein relative to total solids;

at least 95% BLG relative to total protein, wherein said BLG food composition is a dry powder and has a bulk density of at least 0.50 g/mL and preferably at least 0.60 g/mL.

In other preferred embodiments of the present invention, the BLG-based food composition contains no more than 6% w/w. water;

at least 80% total protein relative to total solids;

at least 95% BLG relative to total protein, wherein said BLG-based food composition: is a dry powder;

characterized by a bulk density of at least 0.50 g/ml and preferably at least 0.60 g/ml; and is characterized by a BLG crystallinity of at least 20% and preferably at least 40%.

In additional preferred embodiments of the present invention, the BLG-based food composition contains no more than 6% w/w. water;

at least 80% total protein relative to total solids;

at least 95% BLG relative to total protein, and wherein said BLG food composition:

is a dry powder;

characterized by a bulk density of at least 0.50 g/ml and preferably at least 0.60 g/ml; and is characterized by a degree of protein denaturation of not more than 2% and preferably not more than 1.0%.

In additional preferred embodiments of the present invention, the BLG-based food composition contains no more than 6% w/w. water;

at least 80% total protein relative to total solids;

at least 95% BLG relative to total protein;

not more than 80 mg of phosphorus per 100 g of protein, while the specified food composition based on BLG: is a dry powder.

In still other preferred embodiments of the present invention, the BLG-based food composition contains no more than 6% w/w. water;

at least 90% total protein relative to total solids;

at least 97% BLG relative to total protein;

not more than 50 mg of phosphorus per 100 g of protein, while the specified food composition based on BLG: is a dry powder.

In other preferred embodiments of the present invention, the BLG-based food composition contains no more than 6% w/w. water;

at least 80% total protein based on total solids and preferably at least 90% total protein based on total solids;

30-70% BLG relative to total protein;

8-25% w/w ALA relative to total protein, wherein said BLG food composition is a dry powder and has a BLG crystallinity of at least 20% and preferably at least 40%.

In some preferred embodiments of the present invention, the BLG food composition comprises

20-80% w/w water and preferably 20-60% wt./weight. water;

at least 80% total protein based on total solids, and preferably at least 90% total protein;

- 31 040134 at least 95% BLG relative to total protein;

not more than 80 mg of phosphorus per 100 g of protein, wherein said BLG food composition has a BLG crystallinity of at least 20% and preferably at least 40%, and optionally has a protein denaturation of at most 2% and preferably at most 1 .0%.

The nutritional compositions of these embodiments are particularly useful in preparing dried BLG-based food compositions and are particularly useful for spray drying and producing high density milk whey protein powder with an optimal concentration profile of different milk whey protein species, but containing at least a portion of the BLG in dried BLG crystals.

In other preferred embodiments of the present invention, the BLG food composition comprises

20-80% w/w water and preferably 20-60% wt./weight. water;

at least 80% total protein based on total solids and preferably at least 90% total protein based on total solids;

30-70% BLG relative to total protein;

8-25% w/w ALA relative to total protein, wherein said BLG-based food composition:

characterized by a degree of BLG crystallinity of at least 20% and preferably at least 40%.

The nutritional compositions of these embodiments are particularly useful in preparing dried BLG-based food compositions and are particularly useful for spray drying and producing high density milk whey protein powder with an optimal concentration profile of different milk whey protein species, but containing at least a portion of the BLG in dried BLG crystals.

Another aspect of the present invention relates to the use of a BLG based food composition as defined herein as a food ingredient.

For example, it may be preferable to use the low phosphorus BLG food composition defined herein as a food ingredient in the preparation of a low phosphorus food product.

A further aspect of the present invention relates to a food product containing a BLG-based food composition as defined herein and at least an additional ingredient such as, for example, a source of fats and/or carbohydrates.

In some preferred embodiments of the present invention, the food product is a dry food product, such as a bar containing carbohydrates and proteins, and the specified dry food product contains at least 1% wt./weight. BLG, preferably at least 5%, where:

i) the degree of crystallinity of BLG is at least 20%, preferably at least 40%; and/or ii) at least 90% wt./weight. of the total protein is BLG.

In some particularly preferred embodiments of the present invention, the food product is a low phosphorus food product containing no more than 100 mg of phosphorus per 100 g of protein, preferably no more than 80 mg of phosphorus per 100 g of protein, more preferably no more than 40 mg of phosphorus per 100 g protein and even more preferably not more than 20 mg of phosphorus per 100 g of protein.

BLG has a favorable amino acid profile and preferably provides a significant proportion of the protein content of the food. This is of particular interest if the food product is a food product with a low content of minerals or phosphorus. In some preferred embodiments of the present invention, the BLG-based food composition provides at least 25% w/w. of the total amount of protein in the food product or at least 50% wt./wt., more preferably at least 80% wt./weight. and even more preferably at least 90% wt./weight. It may be even more preferred that the BLG food composition provides all of the protein in the food.

In some preferred embodiments of the present invention, the BLG-based low phosphorus food composition provides at least 25% w/w. of the total amount of protein in the food product with a low content of phosphorus or at least 50% wt./weight., more preferably at least 80% wt./weight. and even more preferably at least 90% wt./weight. It may be even more preferred that the low phosphorus BLG food composition provides all of the protein in the low phosphorus food product.

- 32 040134

Non-limiting examples of a food product are, for example, a dairy product, a candy, a drink, a protein bar, an enteral nutritional composition, a bakery product.

In some preferred embodiments of the present invention, the food product is a drink. The beverage preferably contains a BLG food composition as defined herein providing a total amount of BLG of at least 1% w/w, preferably at least 5% w/w, more preferably at least 8% wt./wt. and even more preferably at least 12% w/w;

a sweetener, such as a sugar sweetener and/or a sugar-free sweetener;

at least one food acid, such as citric acid, or other suitable food acids;

optional flavoring; and not more than 80 mg of phosphorus/100 g of protein, while it is characterized by a pH in the range of 2.5-4.0.

The inventors of the present invention have realized that obtaining acidic, high protein, low mineral beverages or liquids from a BLG based dry food composition containing BLG crystals is a difficult task. A BLG dry food composition containing BLG crystals will typically create a pH range of 5-6 when resuspended in water, while the addition of acids or salts to change the pH or increase conductivity also increases the mineral load in the resulting fluids. drink.

However, the present inventors have found that if a carboxylic acid, lactone, carboxylic anhydride, or a combination thereof is used to lower the pH, then there is no need to add minerals and thereby achieve better control of the composition of minerals in the beverage/liquid.

Thus, one aspect of the present invention relates to a method for producing an acidified, low mineral content liquid using a BLG-based food composition containing BLG crystals as an ingredient, the method comprising the steps of providing one or more acidifying agents selected from the group , consisting of a carboxylic acid, a lactone, a carboxylic anhydride, or a combination thereof, bringing into contact a BLG-based food composition containing BLG crystals with one or more acidifying agents and optionally additional ingredients such as, for example, water, a fat source and/ or a carbohydrate source, wherein said one or more acidifying agents are used in an amount sufficient to adjust the pH to 2-4.5 and preferably 2.5-4.0 and allow the BLG crystals to dissolve, thereby forming a liquid.

For example, the liquid may be used as a beverage, or it may be used as an ingredient in another food product.

If the BLG food composition used in this method is provided in a dry form, such as a powder, it is often preferable to rehydrate it in water before adding the acidifying agent.

The BLG-based nutritional composition used in this method is preferably present in the liquid in an amount sufficient to provide 1-30% w/w. protein, preferably 225% wt./weight. protein, more preferably 4-20% wt./weight. protein and even more preferably 5-16% wt./weight. squirrel.

The BLG food composition used in this method preferably has a BLG crystallinity of at least 30%, preferably at least 50%, and even more preferably at least 70%.

Examples of suitable acidifying agent(s):

carboxylic acids such as, for example, acetic acid, maleic acid, tartaric acid, lactic acid, citric acid, gluconic acid or mixtures thereof;

lactones, such as, for example, D-glucono-delta-lactone;

anhydrides of carboxylic acids.

In some preferred embodiments, the BLG food composition containing BLG crystals used in this method is preferably a low phosphorus composition, with any other ingredients used in this method preferably selected so that the finished liquid is also composition with low phosphorus content.

In other preferred embodiments, the BLG food composition containing BLG crystals used in this method is preferably a low mineral composition, with any other ingredients used in

- 33 040134 of this method are preferably chosen so that the finished liquid is also a composition with a low mineral content.

The method is preferably carried out at a temperature in the range of 1-65°C, preferably 250°C, more preferably in the range of 3-20°C, even more preferably in the range of 4-15°C.

The present invention has been described above with reference to specific embodiments. However, within the scope of the present invention, embodiments other than those described above are equally possible. Various features and steps of various embodiments and aspects of the present invention may be combined in ways other than those described herein, unless otherwise indicated.

Examples

Example 1 Crystallization of β-lactoglobulin from crude milk whey protein concentrate.

Protocol.

Lactose-depleted UF retentate obtained from sweet whey in a standard cheese production process and filtered through a 1.2 μm filter was used as a raw material for a BLG crystallization process. The sweet whey feedstock was processed in an ultrafiltration unit using a HFK-328 Koch membrane with a 46 mil separator, 1.5-3.0 bar supply pressure, using a feedstock concentration of 21% TS (total solids) ±5 and deep demineralized water (water filtered by reverse osmosis to achieve a conductivity of not more than 0.05 mS/cm) as the diafiltration medium. The temperature of the raw material and retentate during ultrafiltration was approx. 12°C. The pH was then adjusted by adding HCl to reach a pH of approx. 5.40. Diafiltration was continued until the drop in the conductivity of the retentate reached a level below 0.03 mS/cm over a period of 20 minutes. The retentate was then concentrated to approx. 30% TS (approx. 23.1% total protein based on the total weight of the concentrated retentate). A sample of the concentrated retentate was centrifuged at 3000 g for 5 minutes, but no visually distinguishable pellets were formed. The supernatant was analyzed by HPLC. The composition of the raw material is shown in table. 1.

The concentrated retentate was seeded at 0.5 g/l, which is pure BLG-based crystalline material resulting from the spontaneous crystallization of BLG (as described in Example 3 in the context of Raw Material 2). Seed material was prepared by washing the suspension of BLG crystals 5 times in milliQ water, collecting the BLG crystals after each wash. After washing, the BLG crystals were freeze-dried, ground with a pestle and mortar, and then passed through a sieve with a pore size of 200 μm. Thus, the seed for crystallization was characterized by a particle size of less than 200 μm.

The concentrated retentate was transferred to a 300 L crystallization tank where it was cooled to about 4° C. and held at that temperature overnight with moderate agitation. The next morning, a sample of the cooled concentrated retentate was transferred to a test tube and examined visually and under a microscope. Rapidly settling crystals clearly formed during the night. A laboratory sample of the mixture containing both crystals and mother liquor was further cooled to 0° C. in an ice water bath. The mother liquor and crystals were separated by centrifugation at 3000 g for 5 min, and the supernatant and pellet samples were taken for HPLC analysis. The crystals were washed once in cold, deeply demineralized water and then centrifuged again before being dried by lyophilization of the granules.

- 34 040134

Table 1. The concentration of certain components of the raw material, standardized to 95% wt./wt. total solids

Raw material standardized to 95% TS

Protein composition (% w/w relative to total protein)

ALA 17.7 BLG 51.6 CMP 19.5

Other components (% w/w relative to the total weight of the standardized raw material)

Ca0.357

K0.200

Mg0.058

Na0.045

P0.280

Fats5,6

Proteins79

Quantification of the relative yield of BLG by HPLC.

All samples were diluted to the same extent by adding deeply demineralized water. The samples were filtered through a filter with a pore size of 0.22 μm. For each sample, the same volume was loaded onto an HPLC system with a Phenomenex Jupiter® 5 µm C4 300 A, 250x4.6 mm LC column (part number: 00G-4167-E0) and detected at 214 nm.

Samples were analyzed under the following conditions:

buffer A: MilliQ water, 0.1% w/w TFA buffer B: HPLC acetonitrile, 0.085% w/w TFA.

Flow rate: 1 ml/min.

Gradient: 0-30 min, 82-55% A and 18-45% B; 30-32 min, 55-10% A and 45-90% B; 32.5-37.5 min, 10% A and 90% B; 38-48 min, 10-82% A and 90-18% B.

Data processing.

Since all samples were treated in the same way, one can directly compare the area of the BLG peaks to obtain relative yield. Since the crystals contain only BLG and all samples were treated in the same way, the concentration of alpha-lactalbumin (ALA) and therefore the area of ALA will be the same in all samples, so the area of ALA before and after crystallization is used as a correction factor (cf) when calculating the relative output _ AREA ABAD ₀ crystallization

AREA ABA _after crystallization

The relative yield is calculated using the following equation:

η А C/α * crystallization area BLG _n0CJie \

Yield \u003d 1------------- x 100 y AREA tiLu ^ ₀ crystallization /

Results.

In FIG. 1 shows overlapping chromatograms before and after crystallization of BLG from sweet whey. The pre-crystallization sample is represented by a solid black line and the post-crystallization sample by a dotted line. It can be seen that there was a significant decrease in the concentration of BLG, and using the yield calculation as described previously, the yield of removed BLG was determined to be 64.5% w/w.

- 35 040134

The crystal suspension was assessed by microscopy; as can be seen in FIG. 2, the sample contained hexagonal crystals, with many having a size well in excess of 200 µm, indicating that the observed crystals are not only seed crystals.

The crystals were easily crushed when squeezed with a needle, which confirmed that they were protein crystals.

In FIG. 3 shows the chromatogram of the washed crystalline product, in which case BLG accounts for 98.9% of the total chromatogram area. The purity of the BLG product can be further improved by additional washing.

Conclusion.

This example demonstrates that it is surprisingly possible to crystallize BLG selectively from a crude whey protein concentrate that contains more than 48% non-BLG protein relative to total protein, and that the resulting BLG crystal isolate is of very high purity. This result opens up a new approach for commercial milk protein separation in which BLG is separated from other protein components in a gentle manner, preferably avoiding prolonged exposure to high temperatures and problematic chemicals.

Example 2 Effect of Conductivity and Temperature on BLG Output.

Protocol.

Using the same raw material, experimental and analytical setups as in Example 1, retentate samples (approx. 13.9% w/w total protein) were taken during ultra-/diafiltration at various levels of conductivity to study the effect of conductivity on crystal yield. BLG. The samples were cooled to 4°C and kept at this temperature overnight (however, the authors found that 30 min or even less may be sufficient to reach equilibrium), and then three samples were cooled to 0°C in ice water and kept at this temperature for at least 1 hour to show the effect of temperature on yield. The results for samples at 4°C can be seen in FIG. 4.

After completion of diafiltration, samples were taken at solids content in degrees Brix of 21, 24 and 32.5 during concentration. These samples were first cooled to 4°C and kept at this temperature overnight. The yield of BLG crystals was measured as described in Example 1. The samples were then cooled to 0° C. in ice water and held at this temperature for at least 1 hour. The yield of BLG crystals was then measured again.

Results:

when plotted on the plot of relative BLG yield versus sample conductivity shown in FIG. 4, there is a clear correlation between lower conductivity and higher relative BLG yield.

In FIG. 5, where the yield for three samples with varying conductivity is shown at two temperatures (4 and 0°C), it can be seen that the lower the temperature, the higher the BLG yield. Lowering the temperature further is expected to increase yield.

In FIG. 6 shows the effect of protein concentration on the relative yield of BLG at both 4 and 0°C. The figure shows a clear correlation between the protein concentration, shown here in degrees Brix, and the relative yield of BLG, indicating that the relative yield continues to increase with increasing protein concentration.

Conclusion.

The authors found that a number of parameters affect the efficiency of the crystallization method. At a given pH, the BLG yield can be increased by lowering the conductivity, increasing the BLG concentration, and lowering the temperature.

Example 3 Crystallization of BLG in three types of milk whey protein solutions.

Protocol.

Using the same experimental and analytical setups as in Example 1, three different types of whey protein-containing raw material were tested as raw material for crystallization. However, in the experiment conducted with raw material 2, no seed was used. Raw materials 1 and 2 are based on sweet whey and have their fat content reduced by a Synder FR membrane prior to treatment as described in Example 1. Raw material 3 is prepared from acid whey.

The composition of the three types of raw material can be seen in the table below. 2-4. Raw material 3 crystallized at 21% TS (total protein 13.3% w/w relative to the total weight of the raw material), i.e. at a significantly lower concentration than the other two (total protein 26.3% w/w in the raw material). material 1 and 25.0% w/w in raw material 2).

The suspension of crystallized raw material 1 was centrifuged on a Maxi-Spin filter with a 0.45 μm CA membrane at 1500 g for 5 min, then 2 volumes of MilliQ water was added to the filter cake prior to subsequent centrifugation. The resulting filter cake was analyzed by HPLC. A photograph of a Maxi-Spin filter with pellets (filter cake) of crystallized raw material 1 is shown in FIG. 24. Raw material 2 pellets were washed with 2 volumes of MilliQ water and centrifuged again under standard conditions prior to analysis of the pellets by HPLC. Raw material 3 pellets were analyzed without washing.

The crystals obtained from raw material 2 were diluted to 10%TS and the pH was adjusted to pH 7 with 1M NaOH to reverse crystallization. NaCl was added to the crystal slurry from Raw Material 2, 36% TS to reverse crystallization.

Table 2. Concentration of Certain Components of Raw Material 1 (Whey Protein Concentrate Based on Sweet Whey) BDL= Below detection level in wet sample

Raw material 1 (standardized to 95% TS) Protein composition (% w/w relative to total protein) ALA 23.0 BLG 55.1 SMR 20.5 Other components (% w/w relative to the total weight of the standardized raw material) Sa 0.387 TO 0.290 mg 0.066 Na 0.068 R 0.207 Fats BDL Protein concentration 90

- 37 040134

Table 3. Concentration of Certain Components of Raw Material 2 (ALA-Reduced Whey Protein Concentrate Based on Sweet Whey) BDL= Below Detection Level in Wet Non-Standardized Sample

Raw material 2 standardized to 95% TS Protein composition (% w/w relative to total protein) ALA 12.2 BLG 70.0 SMR 17.1 Other components (% w/w relative to the total weight of the standardized raw material) Sa 0.387 TO 0.204 mg 0.066 Na 0.051 R 0.174 Fats BDL Protein concentration 89

- 38 040134

Table 4. Concentration of Certain Components of Raw Material 3 (Milk Whey Protein Concentrate Based on Acid Whey)

Raw material 3 standardized to 95% TS Protein composition (% w/w relative to total protein) ALA 24.0 BLG 63.6 Other dairy whey proteins 12.4 Other components (% w/w relative to the total weight of the standardized raw material) Sa 0.205 TO 0.051 mg 0.013 Na 0.108 R 0.240 Fats 5.1 Protein concentration 79

Results. raw material 1.

In FIG. 7, chromatograms of the protein composition of the raw material (solid line) and mother liquor (dashed line) can be seen. Obviously, during this method, most of the BLG was recovered in the form of crystals. The yield (calculated as described in Example 1) of isolated BLG was approx. 65% relative to the total amount of BLG in the raw material.

In FIG. 8 is a microscopy photograph of a sample taken in the early stages of the crystallization period. In FIG. 9 is a microscopy photograph of a sample taken at the end of crystallization. These two images clearly show that BLG crystals are relatively brittle. Some of the crystals appear to break during agitation and change from a hexagonal or rhombic shape to crystal fragments that still appear to be very compact and distinctly structured, but are more irregular in shape.

In FIG. 10 shows a chromatogram of BLG crystals that were separated and washed on a centrifugal filter. This figure shows that the purity is very high and that the removal of other milk whey proteins was particularly effective.

Raw material 2.

In FIG. 11, the protein composition of the raw material 2 (solid line) and the resulting mother liquor (dashed line) can be seen. It appears that most of the BLG was removed and the calculated yield was 82% based on the total BLG in Raw Material 2.

In FIG. 12 shows raw material 2 before (left image) and after (right image) crystallization. During crystallization, the raw material changed from a clear liquid (in which the magnetic stirrer is visible) to a milky white opaque liquid.

- 39 040134

In FIG. 13 shows a microscopy photograph of BLG crystals. The presence of hexagonal forms can be noted, since most of the crystals are fragmented.

In FIG. 16 is a chromatogram of isolated pellets of BLG crystals after washing with volumes of MilliQ water. The chromatogram clearly shows that the crystals contain very high purity BLG.

In FIG. 14 and 15 show the results of either increasing the conductivity (by adding NaCl) or changing the pH (by bringing the pH to 7 by adding NaOH) so that the medium no longer favors the crystal structure. In both cases, the milky white suspension turns to a clear liquid as the BLG crystals dissolve.

The mineral composition of the crystalline preparation obtained from raw material 2 is shown in Table 1. 5. It was noted that the ratio of phosphorus to protein was very low, making the crystalline preparation suitable as a protein source for patients with kidney disease.

Table 5. The concentration of certain components in the crystalline ___________________ preparation obtained from the raw material 2___________________

Composition of the crystalline preparation obtained from raw material 2 % w/w relative to the composition, standardized to 95% TS Sa 0.119 TO 0.047 mg 0.019 Na BDL R BDL (less than 0.026) total protein 93.4

Raw material 3.

In FIG. 17 shows the chromatograms of the protein composition of the raw material 3 (solid line) and the resulting mother liquor (dashed line) are shown. It appears that most of the BLG was recovered (calculated yield was 70.3% of the total BLG in the feed). If the protein content had been higher prior to crystallization, the resulting yield would have been even higher.

In FIG. 18 is a microscopy photograph of BLG crystals isolated from raw material 3 (substantially free of CMP). The crystals were rectangular, not hexagonal. Rectangular crystals looked more durable than hexagonal ones. In FIG. 19 shows a chromatogram of isolated crystal beads without washing; the chromatogram clearly shows that the crystals were BLG crystals despite having a rectangular shape rather than a hexagonal shape (compare, for example, the rectangular crystals in Fig. 18 with the hexagonal crystals in Fig. 2).

- 40 040134

Table 6 Concentration of Certain Components in the Crystal Preparation Obtained from Raw Material 3

Composition of the crystalline preparation obtained from raw material 3 % w/w relative crystal preparation, standardized to 95% TS Sa 0.103 TO BDL mg 0.006 Na 0.035 R 0.041 total protein 90

The crystalline preparation obtained from raw material 3 contained 45 mg P/100 g of protein. It was noted that the ratio of phosphorus to protein was very low, making the crystalline preparation suitable as a protein source for patients with kidney disease.

Conclusion.

All three raw materials were suitable for the BLG crystallization process. The BLG crystals were easily dissolved by adding salt or raising the pH or temperature. The new method makes it possible to obtain BLG-based preparations with very low phosphorus levels, making the preparations suitable as protein sources for patients with kidney disease.

Example 4 Effect of pH on the Yield of BLG Crystals.

Protocol.

The same protocol and experimental setup as in Example 1 was used (using reduced fat sweet whey protein concentrates) except that the pH was adjusted to the levels described in Table 1. 8 for each of the experiments. The protein concentration at the beginning of the crystallization stage was approx. 24% w/w

The pH was adjusted with either a weak NaOH solution (>4%) or a weak HCl solution (>3.6%) to evaluate the effect of the pH on the crystallization process and the yield achieved. After crystallization, BLG crystals were separated by centrifugation as described in Example 1.

- 41 040134

Table 7. Concentration Ranges of Certain Components of the Raw Material Used for Example 4

Raw material used standardized to 95% TS Protein composition, % of total protein (%) ALA BLG CMP Other components (wt% of the total weight of material standards) Sa 10-15 60-70 12-17 :/weight relative to this commodity 0.36-0.45 TO 0.18-0.22 mg 0.04-0.08 Na 0.02-0.06 R 0.17-0.20 Fats BDL Protein concentration 89-91

Table 8. Target pH of Samples

Results.

The yields were calculated as described in example 1. It should be noted that the original samples were taken before adding seed material. Thus, if the samples were not oversaturated with BLG, the seed material would dissolve and provide a total BLG concentration, in which case the BLG yield would be negative.

Table 9. Calculated sample yields based on HPLC measurements

Sample Index pH Yield of total BLG (%) 1 4.84 -2.7 2 5.20 50.0 3 5.44 82.0 4 5.73 62.1 5 5.93 40.9 6 6.12 -1.6

Conclusion.

This experiment showed that BLG crystallization in the salting-in mode is possible in the pH range of 5-6.

EXAMPLE 5 Investigation of the Effect of Increasing Conductivity Levels.

Protocol.

Used the same protocol and experimental setup as in example 1 except that the samples were taken at different conductivity. The crude material shown in table. 10 was processed and used as raw material for the crystallization process. Prior to ultrafiltration, samples of the crude material were taken and NaCl was added to increase the conductivity and to assess at what levels of conductivity the BLG crystals were able to grow. The protein content during crystallization was approx. 16.7% w/w

- 43 040134

Table 10 Composition ranges of the raw material used in Example 5

Raw material used standardized to 95% TS Protein composition (% w/w relative total protein) ALA BLG CMP 12.4 64.5 19.8 Other components (% w/w relative to the total weight of the standardized raw material) Sa TO mg Na R Fats Protein concentration 0.523 0.534 0.085 0.160 0.235 BDL 86

Results.

Samples were processed as described in Example 1. FIG. 20 shows the calculated yields at different retentate conductivities. The point at 3.53 mS/cm was the crude material after pH adjustment. All points above 3.53 were the result of adding NaCl to increase conductivity. Points below 3.53 were the result of diafiltration in the ultrafiltration system. The yield at 4.93 mS/cm was close to zero and was not considered significant.

The retentate sample, which had a conductivity of 4.93 mS/cm, had an ultrafiltrate conductivity of approx. 5.7 mS/cm. The retentate sample with a conductivity of 3.53 mS/cm had an ultrafiltrate conductivity of approx. 4.35 mS/cm.

In FIG. 20 it can be seen that BLG crystals formed in the raw material at conductivity values below 4.93 mS/cm (at 4°C and a total protein content of approx. 16.7% w/w). With a conductivity of the retentate of approx. 2 mS/cm and an ultrafiltrate conductivity of approx. 1.6 mS/cm, a BLG yield of approx. 75% received.

In FIG. 21 shows a microscopic photograph of the crystals formed at 4.20 mS/cm in the retentate, demonstrating the expected characteristics of the BLG crystals.

Conclusion.

The specific raw material from Example 5 allowed BLG crystals to form below 4.93 mS/cm (corresponding to an ultrafiltrate conductivity of 5.75 mS/cm and a conductivity to total protein ratio of 0.057). The upper limit of conductivity is expected to depend on protein concentration and protein composition. For example, a higher protein concentration and/or an increased content of highly charged proteins or other macromolecules (eg, CMP) is expected to increase the upper limit of conductivity at which BLG crystallization is possible.

Example 6 Crystallization of BLG in Whey Protein Concentrate.

Whey Protein Concentrate (SPC) was obtained by processing skimmed milk with

- 44 040134 Microfiltration shaft with Synder FR membrane and operating temperature approx. 50°C. The resulting retentate contained substantially all of the casein and residual fat, and in addition contained some whey proteins, lactose and minerals. The filtrate contained molecules that are able to permeate the membrane, including whey protein, lactose, and minerals, but essentially no casein or fat. The crystallization filtrate was then prepared as described in Example 1 (see Table 11 for the composition of the raw material) and the resulting BLG crystals were characterized as described in Example 1. However, instead of performing all ultrafiltration steps at 12°C, the temperature of the retentate was increased from 12 to 25°C when the conductivity of the retentate reaches 1 mS/cm. The temperature was raised in order to avoid spontaneous crystallization of BLG upon concentration by ultrafiltration.

Table 11. Concentration of Certain Raw Material Components (Whey Protein Concentrate) BDL=Below Detection Level in Wet Non-Standardized Sample

Raw material standardized to 95% TS Protein composition (% w/w relative to total protein) ALA 23.5 BLG 66.7 Other dairy whey proteins 9.8 Other components (% w/w relative to the total weight of the standardized raw material) Sa 0.292 TO BDL mg 0.042 Na BDL R 0.149 Fats BDL Protein concentration 91

Similar to the crystallizations in Examples 1-5, BLG from the SPC feedstock produced high purity crystals that can be separated (confirmed by chromatography as in previous examples) while providing a BLG yield of 70% of total BLG. from the raw material, which is SPC. In FIG. 22 shows BLG crystals obtained in the early stages of crystallization. As shown above, the crystals are characterized by a rectangular or square shape, but not the hexagonal shape observed, for example, in example 2.

Example 7 Preparation of spray dried BLG crystals and determination of bulk density.

A portion of the BLG crystals obtained in Example 3 (using raw material 2) was separated in a decanter centrifuge at 1200 g, 5180 rpm, 110 rpm deviation. with a 64 mil separator (mil means 1/1000 of an inch) and a flow rate of 25-30 l/h. Then the BLG crystal phase was mixed

- 45 040134

1:1 with highly demineralized water and then separated again in a decanter centrifuge at the same settings. The BLG crystal phase was then mixed with deeply demineralized water to obtain its suspension containing approx. 25% dry matter, and with a BLG crystallinity of approx. 80 and then dried on a pilot-industrial spray dryer with an inlet temperature of 180°C and an outlet temperature of 85°C without any preheating. The temperature of the liquid streams before spray drying was 10-12°C. The resulting powder, taken at the outlet, was characterized by a water content of 4.37% w/w.

The crystallinity of BLG in suspension was approximately 90%.

We also successfully separated the BLG crystal suspension and mother liquor in a decanter centrifuge at 350 g, 2750 rpm, 150 rpm Diff. with 64 mil separator and 75 l/h flow rate. The BLG crystal phase was then mixed 1:2 with deeply demineralised water. The BLG crystal phase was then mixed with deeply demineralized water to form a thick slurry therefrom and then dried in a pilot spray dryer under the same parameters as described above.

The bulk density of the spray-dried powder according to Example 9.3 was then measured and compared with that of standard WPI dried using the same equipment. Standard WPI was found to have a bulk density (based on 625 seals) of 0.39 g/mL, a value that is in the upper end of the normal range for WPI powder. However, the spray-dried crystalline BLG formulation had a bulk density of 0.68 g/mL, which is 75% higher than that of standard WPI (see, eg, FIG. 23). This was really unexpected and provided a number of advantages both in terms of logistics and different applications.

Table 12 Concentration of Specific Components of the Spray-Dried BLG Crystal Formulation of Example 7 BDL=Below Limit of Detection

Spray dried powder BLG crystals Protein composition (% w/w relative to total protein) ALA 0.7 BLG 97.4 CMP BDL Other Ingredients (% w/w relative to total weight of BLG crystal powder) Sa 0.118 TO 0.026 mg 0.017 Na BDL R BDL Water 3.8 Protein concentration 94

Then, a sample of the spray-dried BLG crystal preparation was resuspended in cold demineralized water and the BLG crystals were still visible under the microscope. The addition of citric acid or NaCl caused the BLG crystals to dissolve and

- 46 040134 turned an opaque suspension of crystals into a transparent liquid.

The authors found evidence that prolonged heating during the drying step reduces the amount of BLG that is present in crystalline form. Thus, it is preferable that the effect of heat on the BLG crystal preparation is as low as possible.

Conclusion.

This example showed that suspensions containing BLG crystals can be spray dried and that BLG crystals are still present in the resuspended spray dried powder if the heat in the drying step is controlled.

In addition, the authors found that the bulk density of milk whey protein powder, which contains BLG crystals, is significantly higher than that obtained using standard spray drying streams of dissolved proteins. High density powders allow for more cost effective powder packaging and logistics since less packaging material is needed per kilogram of powder and more powder (by weight) can be transported by a specific container or vehicle.

The high density powder also proved to be easier to handle and less loose and dusty during manufacture and use.

Example 8 Low Phosphorus Protein Drink.

Six low phosphorus beverage samples were prepared using the purified BLG product from Example 3 (crystal preparation obtained from Raw Material 3). All dry ingredients were mixed with demineralized water to make 10 kg of each sample and allowed to hydrate for 1 hour at 10°C.

Table 13 Composition of six beverage samples

Ingredient (% weight/weight) drink sample A IN WITH D E F Dried Cleaned BLG from Ex 3, Raw Material 3 5.0 10.0 5.0 10.0 5.0 10.0 Lemon acid Up to pH 3.5 Up to pH 3.5 Up to pH 3.0 Up to pH 3.0 Up to pH 4.0 Up to pH 4.0 sucrose 10.0 10.0 10.0 10.0 10 10 Demineralized water Up to 100% Up to 100% Up to 100% Up to 100% Up to 100% Up to 100%

Aliquots of six samples were taken to measure turbidity on a Turbiquant® 3000 IR turbidimeter and viscosity on a Gilson viscometer. The results are shown in the table below.

Table 14. Measured Viscosity and Haze of Six Beverage Samples

Sample Viscosity (cP) NTU A 1.42 36.2 IN 2.37 46.3 WITH 2.69 4.9 D 2.70 5.0 E 1.45 63.1 F 2.25 82.1

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A photograph of test tubes containing aliquots of six low phosphorus beverage samples is shown in FIG. 25. From left to right, the aliquots were samples A, B, C, D, E, and F. Visual inspection of the test tubes confirmed the turbidity measurements and indicated that all of the beverage samples were clear and that, in particular, samples C and D (pH 3 ,0) were especially transparent. The low viscosity values demonstrated that the beverage samples were easy to drink.

All ingredients used to produce the drink were low in phosphorus and free of unwanted minerals. Thus, the drinks obtained were characterized by a phosphorus content of approx. 45mg P/100g protein and usually had a very low mineral content. Thus, six drinks are suitable for use as protein drinks for patients with kidney disease.

Example 9. Methods of analysis.

Example 9.1. Determination of lactosylated BLG versus non-lactosylated BLG Quantification of lactosylated BLG and native BLG was performed by LC-MS.

Analyzes were performed on a 6410 Triple Quad MS from Agilent Technologies paired with an HP1200 series HPLC also from Agilent Technologies. A Symmetry300™ C18 column (WAT106172: 5 µm solids, column dimensions 2.1 x 150 mm) was used for separation before ionization and protein detection was performed at 214 nm. Before analysis, the samples were filtered through a filter with a pore size of 0.22 microns. All samples were processed twice.

Analyzes were carried out under the following conditions:

HPLC.

Buffer A: 99.9% MilliQ-vand with 0.1% TFA; Buffer B: 9.9% MilliQ-vand, 90% acetonitrile, 0.1% TFA.

Flow rate: 0.3 ml/min.

Gradient.

0-20 min: 85-60% A and 15-40% B,

20-45 min: 60-50% A and 40-50% B,

45-55 min: 0% A and 100% B,

55-70 min 85% A and 15% B.

Loading volume: 40 µl.

The column temperature was set to 60°C.

Mass spectroscopy.

Ions with a mass/charge ratio of 100-2000 were detected and the data obtained were analyzed using the MassHunter Workstation software, ver. B.04.00. Identical particles (masses) were grouped using inversion of convolution of all shapes. Masses from 18 kDa to 20 kDa were subjected to additional study. The original unchanged mass of BLG-A was 18.361 kDa and BLG-B was 18.276, with lactosylation adding 324 Da to the mass of the protein, by evaluating this mass per unit area, up to 5 lactosylations per protein can be detected. Relative quantification was performed by comparing the signal intensity for each mass index, ignoring the unequal ionization of different particles.

Example 9.2. Determination of total protein.

The total protein content (actual protein) in the sample was determined as described below.

1) Determine the total nitrogen in the sample according to ISO 8968-1/2IIDF 020-1/2. Milk. Determination of nitrogen content. Part 1/2. Determination of nitrogen content using the Kjeldahl method.

2) Determined non-protein nitrogen in the sample according to ISO 8968-4IIDF 020-4. Milk. Determination of nitrogen content. Part 4. Determination of the content of non-protein nitrogen.

3) The total amount of protein was calculated as follows:

^(m total nitrogen ^m non-protein nitrogen) x6.38.

Example 9.3. Determination of the density of uncompacted matter and bulk density.

The density of the dry powder was determined as the ratio of the mass and volume of the powder, which was analyzed using a special Stampf volumemeter (ie measuring cylinder) under certain conditions. Density is usually expressed in g/ml or kg/l.

In this method, a dry powder sample is compacted in a measuring cylinder. After a certain number of compactions, the product volume is read and the density calculated.

Three types of density can be determined using this technique:

bulk density, which represents the weight divided by the volume of the powder after it has been transferred to a special measuring cylinder;

uncompacted density which is the weight divided by the volume of the powder after 100 compactions according to the special conditions in this standard;

bulk density, which is the weight divided by the volume of the powder after 625 compactions according to the special conditions in this standard.

- 48 040134

In this method, a special measuring cylinder with a volume of 250 ml, graduated from 0 to 250 ml, weighing 190±15 g (J. Engelsmann A.G. 67059 Ludwigshafen/Rh) and a Stampf volumemeter, for example J.

Engelsmann A.G.

The density of the uncompacted substance and the bulk density of the dried product are determined according to the following procedure.

Preliminary processing.

The sample to be measured is kept at room temperature.

The sample is then thoroughly mixed by repeated rotation and inversion of the container (avoiding the destruction of the particles). The container is no more than 2/3 full.

Procedure.

Weigh out 100.0±0.1 g of the powder and transfer it to a measuring cylinder. The volume V ₀ is determined in ml.

If 100 g of powder does not fit the cylinder, then the amount should be reduced to 50 or 25 g.

Mount a graduated cylinder on a Stampf volumetric meter and carry out 100 seals. Level the surface with a spatula and read the volume V ₁₀₀ in ml.

Switch the number of seals to 625 (incl. 100 seals). After compaction, level the surface and read the volume V ₆₂₅ in ml.

Calculation of density indicators.

Calculate the density of the uncompacted substance and the bulk density, expressed in g/ml, according to the following formula:

bulk density=M/V where M is the weighed sample in grams and V is the volume after 625 compactions in ml.

Example 9.4. Determination of water content in powder.

The water content of the food product was determined according to ISO 5537:2004 (Powdered milk. Determination of moisture content (reference method)). NMKL is short for Nordisk Metodikkomite for Nceringsmidler (Scandinavian Food Analysis Committee).

Example 9.5. Determination of total amounts of calcium, magnesium, sodium, potassium, phosphorus.

The total amount of calcium, magnesium, sodium, potassium and phosphorus is determined using a procedure in which the samples are first destroyed using microwave energy, and then the total amount of mineral substance (s) is determined using the ICP device.

Device.

The microwave sample preparation system is from Anton Paar, and the ICP is the Optima 2000DV from PerkinElmer Inc.

Materials.

1M HNO ₃ , yttrium in 2% HNO ₃ .

Suitable standards for calcium, magnesium, sodium, potassium and phosphorus in 5% HNO3.

Preliminary processing.

Weigh out a certain amount of powder and transfer the powder to a microwave digestion tube. Add 5 ml 1M HNO3. Perform sample digestion in the microwave sample preparation system according to the instructions for microwave sample preparation. Place the digested tubes in a fume hood, remove the lid and evaporate the volatiles.

Measurement procedure.

Transfer the pretreated sample to a Digitube digestion tube using the specified amount of Milli-Q water. Add a solution of yttrium in 2% HNO3 to the digestion tube (approximately 0.25 ml per 50 ml of diluted sample) and dilute to volume using Milli-Q water. Analyze samples for ICP using the procedure described by the manufacturer.

A blank is prepared by diluting a mixture of 10 ml 1M HNO3 and 0.5 ml yttrium in 2% HNO3 to a final volume of 100 ml using Milli-Q water.

Prepared at least 3 standards with concentrations that cover the expected values of the concentration of the samples.

Example 9.6. Determination of the value of furosin content.

Furosin content is determined as described in Maillard Reaction Evaluation by Furosine Determination During Infant Cereal Processing, Guerra-Hernandez et al., Journal of Cereal Science 29 (1999) 171-176, and total protein is determined according to Example 9.2. The value of furosin content is expressed in units of mg furosin per 100 g of protein.

Example 9.7. Determination of the degree of crystallinity of BLG in a liquid.

The following method is used to determine the degree of crystallinity of BLG in a liquid with a pH in the range of 5-6.

a) Transfer a 10 ml sample of the liquid of interest to a Maxi-Spin filter with a 0.45 µm CA membrane pore size.

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b) Immediately centrifuge the filter at 1500 g for 5 min at centrifuge temperature

2°C.

c) Add 2 ml cold milliQ water (2°C) to the retentate side of the centrifugal filter and immediately centrifuge the filter at 1500 g for 5 min keeping the temperature in the centrifuge around 2°C, collect the filtrate (filtrate A), measure the volume and determine the concentration of BLG by HPLC using the method described in example 9.9.

d) Add 4 ml of 2M NaCl from the retentate side of the filter, shake quickly and allow the mixture to stand for 15 min at 25°C.

e) Immediately centrifuge the filter at 1500 g for 5 min and collect the filtrate (filtrate B).

f) Determine the total weight of BLG in filtrate A and filtrate B using the method in Example 9.9 and convert the results to total BLG instead of percent by weight. The weight of BLG in filtrate A is referred to as mf _{ultrat A} and the weight of BLG in filtrate B is referred to as mf _ultra B.

g) The degree of crystallinity of the liquid relative to BLG is defined as crystallinity - P1 _filtrate w / (shf _{filtrate d} + _{Shf filtrate} h) _* 100%

Example 9.8. Determination of the degree of crystallinity of BLG in dry powder.

This method is used to determine the degree of crystallinity of BLG in a dry powder.

a) Mix 5.0 g of powder sample with 20.0 g of cold milliQ water (2°C) and let the mixture stand for 5 min at 2°C.

b) Transfer a sample of the liquid of interest to a Maxi-Spin filter with a 0.45 µm CA membrane pore size.

c) Immediately centrifuge the filter at 1500 g for 5 min at a centrifuge temperature of 2°C.

d) Add 2 ml cold milli-Q water (2°C) from the concentrate side of the centrifugal filter and immediately centrifuge the filter at 1500 g for 5 min, collect the filtrate (filtrate A), measure the volume and determine the BLG concentration by HPLC, using the method shown in example 9.9. and convert the results to total weight BLG instead of percent by weight. The weight of BLG in filtrate A is denoted as mf _I ltrate A.

f) The degree of crystallinity of BLG in the powder is then calculated using the following formula:

^m BLG total ^- ^filtrate A ppp/crystallinity = ----=----— ¹ --*100% ^m BLG total where m _total o _BLG represents the total amount of BLG in the powder sample from step a).

If the total amount of BLG in the powder sample is not known, it can be determined by suspending an additional 5 g of the powder sample (from the same source of powder) in 20.0 g of milliQ water, adjust the pH to 7.0 by adding aqueous NaOH, give the mixture stand for 1 hour at 25°C with stirring, and finally determine the total amount of BLG in the powder sample using example 9.9.

Example 9.9. Determination of the total amount of BLG. ALA and CMP in aqueous liquid.

The contents of alpha-lactalbumin, beta-lactoglobulin and CMP were analyzed by HPLC analysis at 0.4 ml/min. 25 µl of the filtered sample was injected into 2 TSKgel3000PWx1 columns (7.8 mm x 30 cm, Tosohass, Japan) connected in series with an attached PWx1 guard column (6 mm x 4 cm, Tosohass, Japan) equilibrated with the eluent (consisting of 465 g of MilliQ water, 417, 3 g acetonitrile and 1 ml trifluoroacetic acid) and using a UV detector at 210 nm.

Quantification of native alpha-lactalbumin (Salfa), beta-lactoglobulin (C _beta ) and casein macropeptide (C _CMP ) content values was performed by comparing the peak area values obtained for the respective standard proteins with those obtained for the samples.

The total amount of additional protein (non-BLG protein) was determined by subtracting the amount of BLG from the amount of total protein (determined according to example 9.2).

Example 9.10. Determination of the conductivity of the ultrafiltrate.

ml of sample is transferred to an Amicon Ultra-15 centrifuge filter with a molecular weight cut-off of 3 kDa (3000 NMWL) and centrifuged at 4000 g for 20-30 min or until sufficient volume of ultrafiltrate to measure the conductivity accumulates at the bottom centrifugal filters. Conductivity is measured immediately after centrifugation. Processing and centrifugation of samples is carried out at the temperature of the source of the sample.

Example 9.11. Determination of the degree of protein denaturation in a composition based on milk whey protein.

Denatured milk whey protein is known to have lower solubility at pH 4.6 than at pH 7.0, and the degree of denaturation of a milk whey protein composition is determined by measuring the amount of soluble protein at pH 4.6 relative to total amount of protein at pH 7.0.

More specifically, a whey protein composition that is intended

- 50 040134 for analysis (for example, powder or aqueous solution), converted into the first aqueous solution containing 5.0% wt./weight. total protein and characterized by a pH of 7.0; and a second aqueous solution containing 5.0% wt./weight. total protein and characterized by a pH of 4.6.

Adjustment of the pH is carried out using 3% wt./weight. NaOH (aq) or 5% w/w HCl (aq.). The content of total protein (P _p h ₇ ,o) in the first aqueous solution is determined according to example 9.2.

The second aqueous solution is kept for 2 hours at room temperature and then centrifuged at 3000 g for 5 minutes. A sample of the supernatant is removed and analyzed according to example 9.2 to determine the total protein (S _pH4;6 ).

The degree of protein denaturation, D, of the milk whey protein composition is calculated as follows:

D \u003d ((РрН7, О'8рН4, b) / РрН7, o) * 1OO%

Example 9.12. Determination of dried BLG crystals in powder.

The presence of dried BLG crystals in the powder can be determined as follows: the powder sample to be analyzed is resuspended and gently mixed in 4°C demineralized water at a ratio by weight of 2 parts water to 1 part powder, and allowed to rehydrate for 1 hour at 4°C.

The rehydrated sample was visually analyzed under a microscope to determine the presence of crystals, preferably using plane polarized light to detect birefringence.

The crystal-like substance was separated and analyzed by X-ray crystallography to assess the presence of a crystal structure, and also preferably assess whether the crystal lattice (space group and unit cell parameters) corresponds to a BLG crystal.

The chemical composition of the separated crystal-like substance was analyzed to make sure that its solids mainly consisted of BLG.

Example 10 Crystallization by Dynamic Filtration in Tangential Flow Based on Ultrafiltration.

The raw material for the crystallization vessel was prepared as described in example 1, except that the diafiltration was carried out at a pH of 5.92 and the final content of TS was 20%.

After processing the raw material (the composition of the raw material can be seen in Table 15), it was transferred to a 300 L crystallization tank and the pH was initially adjusted to pH 5.80 and the temperature was maintained at 10-12°C. After adjusting the pH, seed material was added, which was obtained in the same manner as described in example 1, but not as a result of spontaneous crystallization. The seed material was added to the raw material at a concentration of 0.5 g of seed material per liter of raw material. After seeding, the temperature of the cooling jacket was set to 5°C, the pH was slowly adjusted to 5.50, and the mixture was left to crystallize for about an hour, after which the DCF (Dynamic tangential flow filtration) unit was connected to the crystallization tank, as shown in fig. 26. The DCF unit was equipped with Kerafol ceramic membranes with a pore size of 500 nm, TMP (transmembrane pressure) was set to 0.4 bar, and the membrane rotation speed was 32 Hz.

The DCF retentate was returned to the crystallization tank while the filtrate was used as feedstock in a UF (ultrafiltration) unit equipped with a HFK-328 Koch membrane with a 46 mil separator. In the UF plant, temperatures were allowed to rise, but to no more than 12°C. The amount of diafiltration water added was adjusted so that the retentate leaving the UF, which flows back into the crystallization tank, contained approximately 21% TS, while minerals were removed from the mother liquor (ML).

The ML diafiltration was continued until the conductivity difference between the filtrate and the diafiltration water was below 50 mS/cm. At this point, the amount of water for diafiltration was adjusted so that the retentate contained approximately 30% TS. The amount of TS in ML decreased when BLG was removed as crystals; this continuous removal of excess water and minerals allows for an increase in overall yield since the concentration of other proteins during BLG crystallization is expected to have a limited effect, if any, on BLG solubility over the ranges that have been investigated.

The composition of the ML filtrate from DCF can be seen in Table 1. 16. The original 300 L of raw material was reduced to about 100 L ML. Based on the conservation of mass, the calculated relative yield of BLG was 92%.

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Table 15. Certain components of the raw material used in example 10

Raw material standardized to

95%TS

Protein composition (% w/w relative to total protein)

ALA 22.9 BLG 50.2 CMP and other proteins 26.5

Other components (% w/w relative to the total weight of the standardized raw material)

Sa 0.387 TO 0.350 mg 0.058 Na 0.245 R 0.210 Fats BDL Squirrels 90

Table 16. Protein composition of the final mother liquor obtained in example 10

Ultimate ML standardized to 95% TS

Protein composition (% w/w relative to total protein)

ALA42.9

BLG6.8

CMP and other proteins50.3

Conclusion:

by continuously removing excess minerals and water from the matrix where the BLG crystallizes, the yield of BLG can be greatly increased while the process can be carried out at low temperatures.

Example 11 Degree of denaturation of proteins from various products based on milk whey proteins.

The degree of protein denaturation was compared from a commercial product and from four BLG food compositions of the present invention.

Samples are described below

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B: suspension of BLG crystals as they are - without drying (present invention)

Samples

A: BiPro (commercially available WPI; Davisco, USA)

C: freeze-dried suspension of BLG crystals (present invention)

D: BLG crystals, re-dissolved (pH 7) and lyophilized

E: spray-dried BLG crystal slurry (present invention)

Samples B-E were prepared as follows: a crystal suspension was prepared as described in example 12 and separated as described in example 7. Some of the separated BLG suspension was taken and divided into four parts.

Sample B: The first part of the separated BLG crystal slurry was redissolved without drying by adjusting the pH of the BLG crystal slurry to 7.01 using 3% NaOH; and then the sample was diluted to Bx 6 to obtain approximately 5% protein solution.

Sample C: The second part of the separated suspension of BLG crystals was freeze-dried. The powder was then resuspended in deeply demineralized water, the pH was adjusted to 7.09 using 3% NaOH, and then the sample was diluted to 6 Brix to obtain an approximately 5% protein solution.

Sample D: The third part of the separated BLG crystal suspension was redissolved by adjusting the pH to 7.0 using 3% NaOH, then lyophilized. The lyophilized powder was then resuspended in deeply demineralized water and the pH measured was 7.07. The sample was then diluted to 6 Brix to obtain an approximately 5% protein solution.

Sample E: A quarter of the separated BLG crystal slurry was treated and spray dried as described in Example 7. The powder was then resuspended in highly demineralized water and the pH was adjusted to 7.04 using 3% NaOH. The sample was then diluted to 6 Brix to obtain an approximately 5% protein solution.

The degree of protein denaturation in each sample was determined according to example 9.11, and the results are presented in table. 17.

Table 17. Comparison of the degree of protein denaturation of a commercially available WPI product (Bipro) with 4 BLG products of the present invention

Sample Total protein concentration at pH 7 General concentration soluble squirrel at pH 4.6 Protein degree denaturation (%) A: BiPro (commercially available WPI) 5.11 4.54 11.15 B: suspension of BLG crystals as they are (without drying) 4.62 4.56 1.30 C: BLG crystal suspension, dried lyophilization 4.74 4.69 1.05 D: BLG crystals, re-dissolved (pH 7) and lyophilized 4.74 4.69 1.05 E: BLG crystal suspension, dried lyophilization 4.75 4.71 0.84

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Conclusion.

Regardless of the drying method, the BLG food compositions of the present invention have an unexpectedly low denatured protein content, which is only a tenth of that measured in the commercially available WPI used for comparison. Particularly surprising is that the spray-dried suspensions of BLG crystals still have the lowest denaturation of any product.

Example 12 Separation of crystals by dynamic filtration in tangential flow.

Lactose-depleted UF retentate obtained from sweet whey in a standard cheese production process, filtered through a filter with a pore size of 1.2 μm, was used as a raw material for a crystallization process. The sweet whey feedstock was processed in an ultrafiltration unit using a HFK-328 Koch type membrane with a 46 mil separator, 1.5-3.0 bar supply pressure, using a feedstock concentration of 10% TS (total solids) ±5 and deep demineralized water (water filtered by reverse osmosis to achieve a conductivity of not more than 0.05 mS/cm) as the diafiltration medium. The temperature of the raw material and retentate during ultrafiltration was approx. 12°C. The pH was then adjusted by adding HCl to reach a pH of approx. 5.60. Diafiltration was continued until the conductivity of the retentate was below 1.30 mS/cm. The feedstock was then heated to 25° C. before concentrating the retentate to approx. 27% TS (approx. 21% total protein relative to the total weight of the concentrated retentate). The conductivity of the filtrate was 0.33 mS/cm at the end of the concentration. A sample of the concentrated retentate was centrifuged at 3000 g for 5 min, but no visually distinguishable pellets were formed.

The concentrated retentate was transferred to a 300 L crystallization tank where it was cooled to about 6° C. and held at that temperature overnight with moderate agitation. By the morning of the next day, the retentate crystallized. The mother liquor and crystals were separated by centrifugation at 3000 g for 5 min, and the supernatant and pellet samples were taken for HPLC analysis. The output of BLG in this way was 67%.

A slurry of crystals from a 300 L tank was used for feedstock in an Andritz DCF 152S system using a single 500 nm pore size membrane disk. Filtration was carried out at 8°C, the rotation speed was 32 Hz, and the transmembrane pressure was 0.4 bar. The system operates as a dead-end filtration where the retentate accumulates in the filter chamber as opposed to large installations where the retentate will be continuously withdrawn. The filtration is carried out in a steady state for only 40 minutes, at which point the solids that have accumulated in the filter chamber begin to affect the filtration. The amount of mass of crystals increased significantly during the DFC process.

Conclusion.

DCF provides a stable and efficient means for separating crystals from ML. If necessary, flushing liquid can be added to the DCF.

Example 13 Separation of crystals using a filtration centrifuge.

Using the same raw material and the same crystallization method as in Example 12, separation was tested in a HZ 25/0.1 filtration centrifuge equipped with a filter cloth with a pore size of about 20 μm.

Test 1:4 L of raw material was fed into a filtration centrifuge which was operated at 60 g. After adding all the raw material, the centrifuge was accelerated to 250 g to dry the filter cake. The precipitate contained 47.6% TS; the composition of the sediment is shown in table. 18.

After purification, 7 liters of the same raw material as described above were fed into the centrifuge at 60 g. The centrifuge was then accelerated to 250 g to remove water for about 5 minutes before speed was reduced again to 60 g, and 0.25 L of deeply demineralized water was added for washing. After adding wash water, the centrifuge was again accelerated to 250 g to remove water. TS in sediment was 47%. The precipitate is shown in Fig. 27A. The composition of the precipitate, fraction ML and washing liquid after washing are shown in table. 18. After dehydration of the sediment, an attempt was made to remove it from the walls of the centrifuge; the top layer decayed and fell out through a special tube, as seen in Fig. 27C and the bottom layer was too wet and sticky to peel off properly, as seen in FIG. 27V.

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Table 18. The concentration of certain components of the compositions presented in example 13

Raw material for AI crystallization ML after centrifugation (Test 1) Filter cake, no rinsing (test 1) ML after centrifugation (Test 2) Filter cake, with washing (test 2) Wash water after washing (test 2) Proteins ¹ / ALA 1.50 1.67 BDL 1.52 BDL 1.07 BLG 12.4 4.10 46.9 5.80 47.9 3.80 CMP 3.56 3.84 BDL 3.3 BDL BDL Other components ²⁾ : Sa 0.410 0.410 0.161 0.529 0.149 0.558 TO 0.315 0.315 0.073 0.424 0.068 0.445 mg 0.075 0.075 0.025 0.098 0.024 0.103 Na 0.078 0.078 BDL 0.109 BDL 0.139 R 0.181 0.181 BDL 0.275 BDL 0.289 total protein 90 90 91 86 93 88

^Protein composition% wt./wt. relative to the weight of the solution.

²⁾ The concentration of other selected components (% wt./wt. relative to the total weight of the composition, standardized to 95% of the total solids).

Conclusion.

Filtration centrifuges provided a promising option for obtaining a cake containing BLG, which is so pure that the content of ALA and CMP is below the level required for quantification even without washing. By using even a small volume of filter cake wash medium, the mineral content of the cake can be further reduced, as can be seen from the protein composition of the wash water in Table 1. 18. The content of non-BLG protein in the precipitate was also reduced by washing, as can be seen using wash water. The wash water used contains ALA and BLG in a ratio that is greater than that in the filter cake. This indicates that the washing step tends to remove ALA (and obviously other non-BLG proteins) more than BLG.

The filter cakes that were obtained in this case were not flaky, but still permeable. This makes it possible to add a drying gas at a given temperature to reduce the moisture content of the filter cake to the point where it becomes flaky like a top layer. Alternatively, the filter cake can be re-dissolved in the centrifuge by adding an appropriate amount of acid, base or salt in aqueous solution in a siphon centrifuge.

Example 14 Influence of the mineral composition of a milk whey protein solution.

In this example, the influence of the molar ratio of monovalent and divalent metal cations on the yield of BLG was investigated.

Two samples were compared.

Sample A: with an excess of Na ⁺ (source: Na ₂ SO ₄ );

Sample B: with excess Ca ²⁺ (source: CaSO ₄ ).

The same type of crude material used in Example 1 was corrected using 2.5% sulfuric acid. The pH of the samples was adjusted to a pH of about 5.4; the exact pH is indicated in the table. 20. The initial volume of each sample was 250 ml. Two samples were dialyzed in another 24 L container against about 24 L of cold deep demineralized water. For all dialysis processes, an OrDial D-Clean MWCO 3500 dialysis tube (part number 63034405) was used. The containers were continuously shaken during the dialysis processes, and the dialysis took place in a cooling device at 4°C. The first dialysis took place during the night.

To remove excess ions after the first dialysis, the dialysis bags were transferred to a container containing 2 L of saline. The concentrations were as follows: sample A: Na ₂ SO ₄ (sodium sulfate) 0.059 M; sample B: CaSO ₄ (calcium sulfate) 0.059M.

The first salt dialysis occurred during the night. Conductivity, pH and solids content on the Brix scale after the first dialysis of salts are shown in table. 20. Saline solutions were replaced with fresh ones and dialysis continued for a week.

After the second salt dialysis, the tubes were transferred to a 24 L container filled with about 24 L of cold, deeply demineralized water and dialyzed overnight to remove excess ions before crystallization.

After the last dialysis step, the protein concentration was somewhat lower than preferred. Thus, the samples were concentrated on a Pellicon XL UF laboratory unit using a membrane with a molecular weight cut-off of 10 kDa and a peristaltic pump operating at 75 ml/h. The content of minerals in the samples together with the crude material are shown in table. 19.

The samples were then seeded with 0.5 g/l of the seed material described earlier and allowed to crystallize at 4° C. overnight. As a result, the next day the crystalline precipitate was visually distinguishable in all samples. Samples for HPLC from each sample were obtained by centrifuging each sample at 3000g for 5 min, followed by analysis of the supernatant sample. The results are shown in table. 21.

Table 19 Concentrations of Certain Mineral Components in Crude Material and Samples A and B from Example 14

Component Calcium Crude material (% w/w) 0.45 Sample A to crist. (% w/w) 0.13 Sample A change ¹ * (%) -71 Sample B to crist. (% w/w) 0.82 Sample B change ¹ ) (%) 82 Chloride 0.21 Not tested Not tested Not tested Not tested Potassium 0.60 0.04 -93 0.06 -90 Magnesium 0.08 0.02 -75 0.02 -75 Sodium 0.14 0.59 321 0.08 -43 Phosphorus 0.22 0.21 -5 0.24 9 Squirrels 86.9 87.8 1 86.2 -1 'Change relative to the concentration of the indicated components in crude material

Table 20. pH value, conductivity and degrees Brix at different stages during the preparation of samples A and B

Sample A Stage pH correction pH value 5.41 The wire. (mS/cm) 3.52 Brix (°) 21.9 A After the first salt dialysis 5.45 6.48 15.4 A After second salt dialysis 5.51 5.76 12.3 A After removal of excess ions by dialysis 5.48 0.883 9.5 A After protein concentration by laboratory ultrafiltration 5.48 1.622 16.4 A Final pH correction 5.42 IN pH correction 5.41 3.52 21.9 IN After the first salt dialysis 5.37 1.55 14.4 IN After second salt dialysis 5.41 1.251 12.4 V After removal of excess ions by dialysis 5.32 0.643 11.3 V After protein concentration by laboratory ultrafiltration 5.32 0.891 11.9 V Final pH correction 5.42

Table 21. BLG concentration in samples with different ratios of mono- and divalent cations

Sample BLG (% w/w)

A (high content of Na ⁺ ) - whey protein solution 6.47 with BLG crystals before separation

A (high content of Na ⁺ ) - mother liquid after 3.94 separation of crystals

B (high Ca ²⁺ content) - whey protein solution 3.56 with BLG crystals before separation

B (high content of Ca ²⁺ ) - mother liquid after 2.22 separation of crystals

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Conclusion.

In table. 21 shows that less residual BLG remains in the mother liquor (and a higher yield of separated BLG crystals is achieved) if a high molar ratio of monovalent to divalent cations is not allowed. The molar ratio of monovalent and divalent cations, and in fact Na + K and Ca + Mg, can be controlled to increase the yield of BLG in the method of the present invention.

Claims (22)

CLAIM 1. A method for producing a food composition containing beta-lactoglobulin (BLG) in crystallized form, the method comprising the step of crystallizing BLG in a supersaturated solution of milk whey protein in a salting-in mode, where said solution of milk whey protein contains BLG and at least one additional milk a whey protein other than BLG, wherein said milk whey protein solution is supersaturated with BLG and has a pH in the range of 5-6; contains BLG in an amount of not more than 85% wt./weight. relative to the total amount of protein, and where the whey protein solution is characterized by a conductivity of not more than 5 mS/cm; or the ratio of the conductivity (expressed in mS/cm) and the total amount of protein in the milk whey protein solution (expressed as wt.% of the total protein relative to the total weight of the milk whey protein solution) is not more than 0.3. 2. The method of claim 1, further comprising the step of separating the BLG crystals from the rest of the milk whey protein solution. 3. The method of claim 2, further comprising the step of washing the BLG crystals. 4. The method of claim 3, wherein the step of washing the BLG crystals comprises washing the separated crystals obtained in the separation step. 5. The method according to any one of claims 2 to 4, further comprising the step of recrystallizing the BLG crystals. 6. The method of claim 5 wherein recrystallizing the BLG crystals comprises recrystallizing the BLG crystals obtained from the separation step or the washing step. 7. The method according to any one of the preceding claims, further comprising the step of drying the BLG-containing composition obtained from the crystallization step, the separation step, the washing step, or the recrystallization step. 8. The method according to any of the previous paragraphs, where the supersaturated solution of milk whey protein contains at least 5% wt./weight. alpha-lactalbumin (ALA) relative to total protein. 9. The method according to any of the previous paragraphs, where the supersaturated solution of milk whey protein contains at least 15% wt./weight. additional milk whey protein other than BLG relative to total protein. 10. The method according to any one of the preceding claims, wherein the milk whey protein solution comprises a whey protein concentrate, a milk whey protein concentrate, a whey protein isolate and/or a milk whey protein isolate. 11. The method according to any one of the preceding claims, wherein the supersaturated whey protein solution is obtained by making one or more of the following adjustments to the whey protein raw material: pH adjustments; decrease in conductivity; lowering the temperature; increase in protein concentration; and adding an agent that reduces water activity. 12. The method of claim 11 wherein the preparation of the milk whey protein solution comprises adjusting the pH of the milk whey protein raw material. 13. The method according to any one of claims 11 or 12, wherein the preparation of the milk whey protein solution comprises reducing the conductivity of the milk whey protein raw material. 14. The method according to any one of claims 11 to 13, wherein the preparation of the milk whey protein solution comprises lowering the temperature of the milk whey protein raw material. 15. The method according to any one of claims 11 to 14, wherein the preparation of the milk whey protein solution comprises increasing the concentration of total protein in the milk whey protein raw material. 16. The method according to any one of the preceding claims, wherein the crystallization of the BLG in the crystallization step comprises one or more of the following: maintaining a supersaturated solution of milk whey protein for a time sufficient for the formation of BLG crystals in the salting-in mode; adding seeds for crystallization; an even greater increase in the values of the degree of supersaturation according to BLG and / or mechanical impact. 17. The method according to any one of claims 2 to 16, where the separation step involves separating BLG crystals to achieve a solids content of at least 30% w/w, preferably at least 40% w/w. and even more preferably at least 50% wt./weight. 18. The method according to any one of claims 3 to 17, wherein the washing in the washing step comprises contacting the separated BLG crystals with a washing liquid without completely dissolving the BLG crystals and then separating the remaining BLG crystals from the washing liquid. 19. The method according to claim 18, where during the washing at the washing stage, no more than 80% wt./weight is dissolved. from the initial number of BLG crystals, preferably not more than 50% wt./weight. and even more preferably not more than 20% wt./weight. from the initial number of BLG crystals. 20. The method according to any one of claims 5 to 19, wherein the recrystallization step comprises dissolving the separated BLG crystals in the recrystallization liquid, adjusting the properties of the recrystallization liquid to achieve BLG supersaturation, wherein said adjustment comprises one or more of the following adjustments: pH adjustment; decrease in conductivity; decrease in temperature; increase in protein concentration; and adding an agent that reduces water activity, crystallizing the BLG in the supersaturated recrystallization liquid with corrected properties, and separating the BLG crystals from the remaining recrystallizing liquid with corrected properties. 21. The method according to any one of claims 5 to 20, wherein the BLG crystals from the washing step are recrystallized at least 2 times. 22. The method according to any one of claims 7 to 21, wherein the drying step comprises one or more of spray drying, lyophilization drying, spin drying, rotary drum drying, and/or fluidized bed drying.